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Traditionalusage,phytochemistryandpharmacologyoftheSouthAfricanmedicinalplantBoophonedisticha(L.f...

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DOI:10.1016/j.jep.2013.10.053·Source:PubMed

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Review

Traditional usage, phytochemistry and pharmacology of the SouthAfrican medicinal plant Boophone disticha (L.f.) Herb. (Amaryllidaceae)

Jerald J. Nair, Johannes Van Staden n

Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01,Scottsville 3209, South Africa

a r t i c l e i n f o

Article history:Received 2 September 2013Received in revised form23 October 2013Accepted 23 October 2013Available online 7 November 2013

Keywords:AlkaloidAmaryllidaceaeBoophone distichaTraditional medicine

a b s t r a c t

Ethnopharmacological relevance: Boophone disticha is the most common member of the South AfricanAmaryllidaceae used extensively in traditional medicine of the various indigenous population groups,including the Sotho, Xhosa and Zulu as well as the San. This survey was carried out to identify andhighlight areas relevant to the traditional usage of Boophone disticha. Pharmacological aspects wereexamined with the purpose of reconciling these with the traditional usage of the plant. In relation tophytochemical make-up, particular attentionwas paid on how its alkaloid constitution might corroboratethe various biological effects manifested by the plant.Materials and methods: Information gathering involved the use of four different database platforms,including Google Scholar, ScienceDirect, SciFinders and Scopus. Arrangement and detailing of thisinformation is as reflected in the various sections of the paper.Results: Sixteen categories were identified under which Boophone disticha finds use in traditionalmedicine. These were shown to include general usage purposes, such as ‘cultural and dietary’, ‘well-being’, ‘personal injury’, ‘divinatory purposes’, ‘psychoactive properties’ and ‘veterinary uses’. Further-more, traditional usage was seen to involve six body systems, including functions pertaining to thecirculatory, gastrointestinal, muscular, neurological, respiratory and urinary systems. The four remainingcategories relate to use for inflammatory conditions, cancer, malaria and tuberculosis. Overall, three areaswere discernible in which Boophone disticha finds most usage, which are (i) ailments pertaining to theCNS, (ii) wounds and infections, and (iii) inflammatory conditions. In addition, several aspects pertainingto the toxic properties of the plant are discussed, including genotoxicity, mutagenicity and neurotoxicity.Conclusion: The widespread ethnic usage of Boophone disticha has justified its standing as a flagship forthe Amaryllidaceae and its relevance to South African traditional medicine. Furthermore, its promisingpharmacological and phytochemical profiles have stimulated significant interest in the clinical realm,especially in the areas of cancer and motor neuron disease chemotherapy. These collective propertiesshould prove useful in steering the progress of the plant towards a wider audience.

& 2013 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132. Description and distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143. Taxonomy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154. Conservation status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155. Traditional usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156. Phytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

6.1. Alkaloids of the South African Amaryllidaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/jep

Journal of Ethnopharmacology

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jep.2013.10.053

Abbreviations: AChE, acetylcholineserase; AD, Alzheimer's disease; BW, body weight; CNS, central nervous system; COX, cyclo-oxygenase; IK, indigenous knowledge;LD, lethal dose; MAO, monoamine oxidase; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; MND, motor neuron disease; MTD,minimum toxic dose; OFS, Orange Free State; PD, Parkinson's disease; SSRI, selective serotonin reuptake inhibitor; T/C index, ratio of treated and control group; TM,traditional medicine

n Corresponding author. Tel.: þ27 332605130.E-mail address: rcpgd@ukzn.ac.za (J. Van Staden).

Journal of Ethnopharmacology 151 (2014) 12–26

6.2. Alkaloids from Boophone disticha . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

7.1. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197.2. Anti-inflammatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207.3. Effects on the central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

7.3.1. Hallucinogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207.3.2. Mental disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

7.4. Anticancer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227.5. Toxicological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

1. Introduction

The Amaryllidaceae J. St.-Hil. is a large family of bulbousflowering plants comprising roughly 1000 species in 79 generawith a pantropical distribution (Meerow and Snijman, 1998).Andean South America, the Mediterranean and South Africa havebeen identified as centres of prominence for these monocotyle-donous plants (Meerow and Snijman, 1998). Their sphere ofinfluence extends into areas as diverse as agriculture, horticulture,ethnobotany, traditional medicine (TM) and pharmacology(Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013). Forexample, daffodils (Narcissus) are amongst the most commonlysold cut-flower varieties and are thus of significant importance tothe floriculture industry (Bastida et al., 2006). Furthermore,several members of the family have intensive usage in thetraditional medicinal practices of indigenous peoples around theworld (Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013).The long history of association of the Amaryllidaceae with TM inthe Mediterranean basin can be traced back to the times ofHippocrates and Pliny who recorded diverse medicinal applica-tions of Narcissus (Pettit et al., 1993; Kornienko and Evidente,2008). Furthermore, archaeological findings at purportedly Incaruins in South America have indicated that Ismene, Pyolirion andStenomesson as depicted on ceremonial drinking vessels may havebeen a part of popular culture (Vargas, 1981), while San rockpaintings of Boophone and Brunsvigia species in South Africa attestto the usage of the Amaryllidaceae by the region's first inhabitants(Dyer, 1950; Van Wyk, 2008).

Roughly one-third of the Amaryllidaceae is found in SouthAfrica, the majority of which are distributed through the Capensisfloral kingdom of the Western Cape (Meerow and Snijman, 1998).Biosystematically, members of the South African Amaryllidaceaeare adherent almost exclusively to the tribes Amaryllideae, Hae-mantheae and Cyrtantheae which are three of the 14 recognisedtribes in this family (Olivier, 1980; Snijman and Linder, 1996;Meerow and Clayton, 2004). Amaryllideae J. St.-Hil. is a mono-phyletic group consisting of 11 currently recognised genera andapproximately 155 species (Snijman and Linder, 1996). Members ofthe tribe, typified by Crinum, inhabit grassland, savannah andtropical forests in sub-Saharan Africa, but are most speciose inSouth Africa where habitats include semi-arid dwarf shrublands inthe winter rainfall region (Snijman and Linder, 1996). By contrast,the tribe Haemantheae Pax (Hutchinson) consists of five genera(Scadoxus, Haemanthus, Clivia, Gethyllis and Apodolirion) which aredistributed through the various regions in South Africa (Meerowand Clayton, 2004). Of the African clades, Cyrtantheae Salisb. isrecognised as a tribe unto itself consisting of a single endemicgenus, Cyrtanthus (Olivier, 1980).

Apart from its morphological and floral characteristics, theAmaryllidaceae is widely known for its unique alkaloid constitu-ents (Viladomat et al., 1997; Bastida et al., 2006; Jin, 2011; Nair

et al., 2013). Chief amongst these secondary metabolites is thealkaloid galanthamine 1 (also referred to as ‘galantamine’)(Scheme 1), a prescription drug in the treatment of Alzheimer'sdisease (AD) (Greig et al., 2004; Heinrich and Teoh, 2004;Houghton et al., 2006). Having captured a significant share ofthe multi-billion dollar global market over the past decade,galanthamine, like other acetylcholinesterase (AChE) inhibitorregimens, remains steadfast as a primary choice in the chemother-apeutic approaches to AD (Greig et al., 2004; Heinrich and Teoh,2004; Houghton et al., 2006). This status is due chiefly to itspotent, selective and reversible inhibitory interaction with AChEwhich is known to play a significant role in the progression ofneurodegeneration associated with the pathophysiology of AD(Greig et al., 2004; Heinrich and Teoh, 2004; Houghton et al.,2006). The research and development of galanthamine into aviable contemporary AD drug owes much to TM knowledgeevolving out of the usage of Galanthus species, from whichgalanthamine was first isolated, in the Caucasus region of EasternEurope (Heinrich and Teoh, 2004).

In terms of structural diversity, Amaryllidaceae alkaloids havebeen classified into six distinct groups (Viladomat et al., 1997;Bastida et al., 2006; Jin, 2011; Nair et al., 2013). Galanthamine 1,lycorine 2 and crinine 3 are representative of the three majorstructural-types for these alkaloids (Scheme 1), while homolycor-ine 4, tazettine 5 and montanine 6 make up the minor series ofcompounds discernible within the Amaryllidaceae (Viladomatet al., 1997; Bastida et al., 2006; Jin, 2011; Nair et al., 2013). Otherless-conspicuous members include degraded, oxidised and trun-cated variants such as trisphaeridine 7 and ismine 8 (Viladomatet al., 1997; Bastida et al., 2006; Jin, 2011; Nair et al., 2013). Allthese structures are related as a consequence of their biogenesisfrom the common amino acid derived precursor norbelladine 9(Viladomat et al., 1997; Bastida et al., 2006; Jin, 2011; Nair et al.,2013).

In addition to the therapeutic success of galanthamine in themotor neuron disease (MND) arena, other clinical candidates havebeen identified in the Amaryllidaceae with significant potential fordrug development. Of these, the phenanthridones (such as pan-cratistatin 10) of the lycorine 2 series have attracted most interestas anticancer agents due to their potent and cell line-specificantiproliferative activities and it has been suggested that acommercial target should be made available within the nextdecade (Kornienko and Evidente, 2008). Thus, with galanthamine1 and pancratistatin 10 entrenched as veritable leads, the Amar-yllidaceae provides a vast resource platform for drug discovery inthe cancer, MND and allied disciplines (Viladomat et al., 1997;Bastida et al., 2006; Nair et al., 2013).

Given these interesting biological traits, it is not surprising thatthe Amaryllidaceae is one of the most common bulbous plantsexploited on a global scale in both traditional and mainstreamsectors of healthcare (Viladomat et al., 1997; Bastida et al., 2006;

J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 13

Fennell and Van Staden, 2001; Nair et al., 2013). This is also truefor the local scene where Amaryllidaceae plants find heavy usagein TM and are consistently amongst the most popular bulbousplants traded on such markets (Hutchings et al., 1996; Louw et al.,2002; Brueton et al., 2008). Amongst the South African Amarylli-daceae contingent, Boophone disticha stands out as the mostcommon plant used in both the formal and informal sectors ofTM, as well as in local research initiatives (Hutchings et al., 1996;Dold and Cocks, 2002; Louw et al., 2002; Brueton et al., 2008).However, the plant is also known to be one of the several toxicspecies of the Amaryllidaceae (Van Wyk et al., 2002, 2005). Thus,within the context of South African TM details of the traditionaluses, phytochemistry and pharmacology of Boophone disticha areafforded herein with the aim of reconciling the traditional aspectsof its usage with more recent discoveries in the pharmacology ofthe Amaryllidaceae.

2. Description and distribution

The genus Boophone Herb. comprises of only two species:Boophone disticha (L.f.) Herb. and Boophone haemanthoides F.M.Leight. (Meerow and Snijman, 1998). Boophone disticha has atvarious instances previously been referred to as ‘Amaryllis dis-ticha’, ‘Boophane disticha’, ‘Boophone toxicaria’, ‘Buphane disticha’and ‘Haemanthus toxicarius’ (Wrinkle, 1984; Meerow and

Snijman, 1998; Williamson, 2012). Now exclusively referred toas Boophone disticha, it is distributed along East Africa, rangingfrom Sudan in the north to the Western Cape Province in thesouth. In South Africa it is widely occurring with a range coveringthe northern tropical savannah woodlands to the winter rainfallregion of the Western Cape, including the desert-like Karoobiome as well as the Eastern seaboard (Wrinkle, 1984). Bycontrast, Boophone haemanthoides is a rare and threatenedspecies with a restricted territory within the winter rainfallregion of South Africa and parts of southern Namibia (Wrinkle,1984). The generic name ‘Boophone’ is derived from the Greek‘bous’ (ox) and ‘phone’ (death) while the specific name ‘disticha’refers to the leaves which are erect in a fan shape. Also known as‘sore-eye flower’ (‘seerooglelie’ in Afrikaans or ‘incotho’ inXhosa), Boophone disticha is an attractive, deciduous plant whichflowers between July and October (Wrinkle, 1984). The largebulbs are covered with a thick layer of papyraceous scales, theskeletal remains of long dried leaves, which rise above soil leveland serve to protect the developing flower and newly emergedleaves (Williamson, 2012). Individual leaves twist slightly alongtheir length and are greyish-green to dark green in colour(Williamson, 2012) (Fig. 1). The peduncle is stout and carries adense, rounded ball of numerous red-pink to dark pink flowerswith long pedicels which elongate even further and stiffen duringmaturity to carry the dried seed capsules (Williamson, 2012)(Fig. 1).

Scheme 1. Structurally diverse and biologically significant alkaloid representatives of the Amaryllidaceae derived from the common biosynthetic precursor norbelladine 9via phenolic oxidative coupling.

J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–2614

3. Taxonomy

Phylogenetic relationships within the Amaryllidaceae havecome under intense scrutiny over the past several decades andsignificant efforts have been directed towards the African tribesAmaryllideae, Haemantheae and Cyrtantheae (Traub, 1963; Olivier,1980; Dahlgren et al., 1985; Müller-Doblies and Müller-Doblies,1996; Snijman and Linder, 1996; Meerow and Snijman, 1998;Meerow et al., 1999; Meerow and Snijman, 2001; Meerow andClayton, 2004). As early as 1963, Traub proposed a two-tierstructure for the tribe Amaryllideae based on both vegetativeand floral morphological characteristics. Accordingly, subtribeCrineae (Ammocharis Herb., Boophone Herb., Brunsvigia Heist.,Crinum L., Cybistetes Milne-Redh. & Schweick. and Nerine Herb.)was distinguished from subtribe Strumarieae (Carpolyza Salisb.,Hessea Herb. and Strumaria Jacq.). Contrary to this subtribalclassification, Dahlgren et al. (1985) combined all nine generalisted above together with Amaryllis L. into the single tribeAmaryllideae. The 1996 classification of Müller-Doblies andMüller-Doblies re-introduces the subtribal structure to the tribeAmaryllideae but in this case with four component subtribes:(i) Amaryllidinae (Amaryllis, Namaquanula D. and U. M-D. andNerine); (ii) Boophoninae (Boophone, Brunsvigia and CrossyneSalisb.); (iii) Strumariinae (Bokkeveldia D. and U. M-D., Carpolyza,Dewinterella D. and U. M-D., Gemmaria D. and U. M-D., Hessea,Strumaria and Tedingea D. and U. M-D.); and (iv) Crininae (Crinum,Ammocharis and Cybistetes). The revision of the tribe Amaryllideaeby Snijman and Linder (1996) is widely accepted as the mostcomprehensive classification to date and reverts back to the initialtwo-tier structure of Traub (1963) with minor adjustments. Basedon morphological, anatomical and cytological data, the tribe wasresolved into two monophyletic subtribes: (i) Crininae comprisingAmmocharis, Boophone, Crinum and Cybistetes; and (ii) Amaryllidi-nae comprising Amaryllis, Brunsvigia, Carpolyza, Crossyne, Hessea,Nerine and Strumaria (Snijman and Linder, 1996). Thus, based onthese characteristics as well as evidence gleaned from DNAsequence data (Meerow and Snijman, 2001), the placement ofthe genus Boophone in the tribe Amaryllideae has never beenin doubt.

4. Conservation status

Given the natural abundance of the Amaryllidaceae in SouthAfrica, its widespread usage in TM, its ornamental value as well as

its medicinally significant alkaloid constituents, it is not surprisingthat these plants feature prominently amongst the manifoldchallenges posed to the conservation sector. This has been furtherexacerbated by more recent concerns such as unsustainable usage,unscrupulous collection methods as well as habitat destruction.A recent review of red-listed medicinal plants of South Africa hasshown that 42 species of the Amaryllidaceae have been documen-ted in TM, representing 2% of the total medicinal flora (Williamset al., 2013). However, of greater concern is that 26% of the totallocal Amaryllid population has been classified as ‘threatened’ or‘near threatened’ (Williams et al., 2013). The status of Boophonedisticha has been indexed as ‘declining’ (Williams et al., 2013)which is not unexpected given the extent of its exploitation in TM(Dold and Cocks, 2002). To this end, advances in seed andmolecular biology as well as the biotechnology arena have expe-dited the availability of propagated plants at TM markets to bolsterstocks under threat from unsustainable practices (Cheesman,2013).

5. Traditional usage

The fact that Boophone disticha can be traced back via rockpaintings to the original inhabitants of South Africa suggests a longhistory of association of the plant with the traditional practices ofthe indigenous peoples of the region (Van Wyk, 2008). This isfurther substantiated by the discovery of the �2000 year-oldremains of a Khoi-San inhabitant of the Kouga/Baviaanskloof areaof the Eastern Cape, found mummified with Boophone distichascales (Binneman, 1999; Van Wyk, 2008). Furthermore, records ofusage of the plant by Cape-Dutch settlers (Watt and Breyer-Brandwijk, 1962) suggest that the application of indigenousknowledge (IK) may have been essential to the medicinal needsof early settlers to the colony. ‘Khoi-San’ is a unifying name for the‘Khoi’ and ‘San’ tribes of southern Africa, two ethnic groups whoshare physical and linguistic characteristics distinct from the Bantumajority of the region (Barnard, 1992). Usage of the Amaryllida-ceae for medicinal purposes in South Africa is extensive which isreflected by the popularity of these plants on traditional marketsof the three most populous tribes of the region (the Sotho, Xhosaand Zulu) (Hutchings et al., 1996; Dold and Cocks, 2002; Louwet al., 2002). For example, Hutchings et al. (1996) documented theusage of 13 different Amaryllid species by traditional healers fromvarious districts within the Zululand area. Of these, eight were forurinary or venereal diseases; six for headache and fever; six for

Fig. 1. Display showing the aesthetic floral and leaf characteristics of Boophone disticha.

J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 15

swelling, growths and joint ailments; five for skin conditions,bruises, sprains and fractures; four for respiratory problems; threefor gastrointestinal disorders; and two for use as internal purifiers(Hutchings et al., 1996). Furthermore, a survey by Dold and Cocks(2002) covering six urban centres in the Eastern Cape Provinceshowed that a minimum of 166 medicinal plants were traded onthese markets of the indigenous Xhosa, utilising 525 tonnes ofplant material with an estimated value of $3 million annually. Inthe study, Boophone disticha was listed amongst the most fre-quently traded plants at these centres (Dold and Cocks, 2002).Another study revealed that Boophone disticha is one of the mostcommonly traded bulbous plants at the Faraday ‘umuthi’market inJohannesburg (the largest TM market in South Africa) (Bruetonet al., 2008). Given these facts, it is clear that Boophone disticha isthe most common of the Amaryllidaceae members exploitedacross many sectors of TM in South Africa.

For the purpose of this survey, 16 categories (Table 1) wereidentified under which Boophone disticha finds use in the TMpractices of indigenous people of the region. Furthermore, whilebulbs were the most common plant part used in TM, leaves,flowers and roots were also utilised but to a lesser extent. Inbroad terms, Boophone disticha is used for cultural (such asadornment), health and well-being purposes (bulb tonic takenfor weakness) as well as for personal injury (wounds, cuts andbruises) (Table 1, entries 1–3). Six categories relating to bodysystem functions were identified, including usage for circulatory,gastrointestinal, muscular, neurological, respiratory, and urinaryailments (Table 1, entries 4–9). Of significance in these categoriesis the use of bulb scales for treatment of asthma (Watt and Breyer-Brandwijk, 1962; De Beer and Van Wyk, 2011) and the use of bulbinfusions for anxiety, depression and age-related dementia (Risaet al., 2004a; Pedersen et al., 2008; Stafford et al., 2008; Neergaardet al., 2009; Risa et al., 2011b). Interestingly, use of the plant forrespiratory ailments was shown to include both indigenous andEuropean respondents highlighting the cross-cultural significanceof the plant. Apart from this Boophone haemanthoides, the onlyother member of the genus Boophone is also utilised for respira-tory illnesses (De Beer and Van Wyk, 2011; Nair et al., 2012b). Thewidespread usage of Boophone disticha for neurological disordersis well documented and further details of this will be provided inSection 7 of this paper. In relation to specific diseases, Boophonedisticha is applicable for treatment of various inflammatory con-ditions, cancer, malaria and tuberculosis (Table 1, entries 10–13).Details of these functions, especially in cancer chemotherapy, willalso be provided in Section 7 of this paper. The three remainingcategories pertain to psychoactive, divinatory and veterinaryproperties in which the plant finds common usage for therapeuticpurposes.

Of the manifold uses of Boophone disticha, its usage as an arrowand dart poison by San communities is most striking (Watt andBreyer-Brandwijk, 1962; Verzár and Petri, 1987; Bisset, 1989; DeSmet, 1998). Amongst the varied purposes for which plant toxinshave come to be used, their primary function as an auxiliaryweapon in the procurement of food has never been disputed(Bisset, 1989; Philippe and Angenot, 2005). In addition, thesesubstances have been widely employed in tribal warfare, as itemsin inter-tribal trade and as ingredients or sources of medicines(Bisset, 1989; Philippe and Angenot, 2005). In present times it isbelieved that this practice still continues within some factions ofthe San people scattered through parts of Namibia and Botswana(Bisset, 1989; De Smet, 1998). Moreover, it is known that the Khoi-San and certain Bantu tribes also once used Boophone disticha forsimilar purposes, but it is actively contended that the practicedescended down from the San (Bisset, 1989; De Smet, 1998). TheKhoi-San used bulbs to poison arrows intended for shootingsmaller types of game, and they appear to have had a profound

knowledge of the bulbs in selecting as more toxic those growingunder cover of shade over those in sunnier spots (Watt and Breyer-Brandwijk, 1962). Evidence for this essential cultural practiceamongst the San can be seen in arrow heads, known to have beentreated with Boophone disticha and taken from the Cape to Berlinin 1806, from which a substance (“haemanthine”) was isolatedover one hundred years later (Lewin, 1912a,b; Watt and Breyer-Brandwijk, 1962). Although no trace of the compound remainedfor comparative studies several decades later, it was nonethelessreputed to be either buphanamine 11 or nerbowdine 12 (Bateset al., 1957; Goosen and Warren, 1957; Fales and Wildman, 1961),two known alkaloid constituents of Boophone disticha (refer toSection 6.2). Furthermore, an arrow belonging to the San of theKalahari, known to have been in possession of the VölkerkundeMuseum der Universität Göttingen since 1937, was shown tocontain alkaloids characteristic of Boophone disticha 60 years later(Mebs et al., 1996; De Smet, 1998). An in-depth look at thephytochemical makeup of Boophone disticha follows in Section6.2 of this paper.

Another area that Boophone disticha finds extensive usage is inthe practice of circumcision and initiation rituals (Table 1) of thevarious indigenous tribes of South Africa (Watt and Breyer-Brandwijk, 1962; Verzár and Petri, 1987; De Smet, 1996;Hutchings et al., 1996; Viladomat et al., 1997; Grierson andAfolayan, 1999; Du Plooy et al., 2001; Van Wyk et al., 2008;Philander, 2011). Male circumcision is performed on adolescentor young men for cultural reasons, particularly as an initiationritual and a rite of passage into manhood (Wilcken et al., 2010). Itis a practice common in Sotho, Xhosa and Zulu culture carried outin a non-clinical setting by a traditional healer (an ‘Ngcibi’) withno formal medical training (De Smet, 1996; Foden, 2010; Wilckenet al., 2010). In the townships of Gauteng Province, 10% of malesaged 14–24 years and 22% of those aged 19–29 years werereportedly circumcised, in 58–65% of cases by traditional circum-cisers (Lagarde et al., 2003). In the same study, it was shown thatchoice of providers depended on the affiliation to different ethnicgroups; for example, 86% of Xhosa participants were circumcisedby traditional healers compared with 37% of Tswana men. Duringthe initiation period, Xhosa boys are covered from head to toewith sandstone which gives them the characteristic white colourand live together in a circumcision lodge under strict conditionsof the lodge master who also instructs them in matters relating tosocial conduct, duty, tradition and political obligation (Foden,2010). In Sotho tradition, initiates are given food mixed withbulbs of Boophone disticha together with other ingredients whichis thought to imbue them with the qualities of their ancestors aswell as to make men out of them (De Smet, 1996). The signs ofintoxication are regarded as a token that the spirit of manhoodhad entered their bodies (De Smet, 1996). Furthermore, bulbinfusions are used by Sotho and Xhosa boys as an outer dressingfor circumcision wounds (Watt and Breyer-Brandwijk, 1962;Verzár and Petri, 1987; De Smet, 1996; Hutchings et al., 1996;Viladomat et al., 1997; Grierson and Afolayan, 1999; Du Plooyet al., 2001; Van Wyk et al., 2008; Philander, 2011). Morerecently, there have been concerted efforts to popularise thehealth benefits of medical male circumcision due to its protectiveeffect in model studies of female-to-male transmission of HIV(Wilcken et al., 2010). This not withstanding, traditional malecircumcision is not without complications and numerous casesare reported on an anual basis relating to excessive bleeding,infection, delayed wound healing and even death (Wilcken et al.,2010). Given these facts, the popularity of the practice on thecultural landscape as well as the recent availability of malecircumcision clinics nationwide, there has been ongoing debateabout the integration of this sector of TM into mainstreamhealthcare in South Africa.

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Table 1Ethnopharmacological usage of Boophone disticha by various indigenous South African groups in the practice of traditional medicine.

Category of use Description of traditional usage References

1. Cultural anddietary

(a) Bulbs used by Sotho and Xhosa boys as an outer dressing for circumcisionwounds

(b) Outer layer of bulbs used to fashion head ring of Swati chief and headman(c) Leaves are stripped to make fringes which are worn as decorative body

ornaments(d) Bulbs were used as an arrow and dart poison by San and Xhoi-San of the Cape(e) Bulbs known to have caused death by suicide in the OFS(f) Prior to tin utensils, scooped-out bulb was used by Sotho herd boys to warm

milk(g) Zulu use bulb scales to plug ear lobes after piercing(h) Cape-Dutch settlers used a sleeping mattress filled with bulb scales to relieve

insomnia(i) Chewed dried leaves used to treat alcohol addiction in the Cape(j) Zulu take flower infusions with porridge for dizziness

Hutchings et al. (1996), Nair et al. (2013), Viladomat et al.(1997), Watt and Breyer-Brandwijk (1962)Watt and Breyer-Brandwijk (1962)Hutchings et al. (1996), Watt and Breyer-Brandwijk (1962)Bisset (1989), De Smet (1996, 1998), Neuwinger (1994),Van Wyk et al. (2002, 2005), Watt and Breyer-Brandwijk(1962)Watt and Breyer-Brandwijk (1962)Watt and Breyer-Brandwijk (1962)Hutchings et al. (1996)Van Wyk et al. (2002), Watt and Breyer-Brandwijk (1962)Philander (2011)Hutchings et al. (1996)

2. Well-being (a) Dry bulb scales taken for feeling of weakness(b) Fresh bulb water extract is drunk to increase sexual potency(c) Bulbs used to improve memory

Verzár and Petri (1987)Verzár and Petri (1987)Risa et al. (2004a, 2004b), Stafford et al. (2008)

3. Personal injury Bulbs are used by most tribes of South Africa (as well as early European settlers tothe Cape) for rashes, bruises, burns, cuts, wounds, boils and swelling. The treatmentis thought to relieve pain as well as to draw out pus

Cheesman et al. (2012)Grierson and Afolayan (1999)Hutchings et al. (1996)Mabona and Van Vuuren (2013)Van Wyk et al. (2002, 2005)Watt and Breyer-Brandwijk (1962)

4. Gastrointestinal (a) Leaves are ingested as an internal wash(b) Bulb decoctions are taken as an emetic and purgative(c) Bulbs used as enemas by the Sotho, Xhosa and Zulu(d) Fresh root and bulb decoction used for constipation

Philander (2011)Hutchings et al. (1996)Hutchings et al. (1996)Wintola and Afolayan (2010)

5. Urinary Bulb concoction taken to purify the bladder and uterus Hutchings et al. (1996)Philander (2011)Viladomat et al. (1997)

6. Circulatory (a) Bulbs used to purify blood(b) Leaves used to stop bleeding

Hutchings et al. (1996)Philander (2011), Viladomat et al. (1997)

7. Respiratory (a) Papery bulb scales used for asthma by Zulu and San(b) Bulbs used for dyspnoea(c) Zulu women roll snuff on a piece of dried bulb scale to improve the efficay of

the snuff

De Beer and Van Wyk (2011), Van Wyk et al. (2008) andWatt and Breyer-Brandwijk (1962)Watt and Breyer-Brandwijk (1962)Watt and Breyer-Brandwijk (1962)

8. Muscular (a) Bulb decoctions are applied for muscle pain and stiffness(b) Oral bulb decoction given to Zulu patients suffering from ‘inkwatshu’,

characterised by cramp-like pain in the calf muscles and tightness in thefingers and toes

Viladomat et al. (1997), Watt and Breyer-Brandwijk (1962)Hutchings et al. (1996), Viladomat et al. (1997)

9. Neurological (a) Roots are burned, ground and the powder applied to the area experiencingparalysis

(b) An old European remedy for hysteria from the Touws River area involvedsleeping on a mattress filled with bulb scales

(c) Bulbs from the Umtentweni area of the KwaZulu-Natal south coast are used inthe treatment of ‘fufunya’ (a type of hysteria)

(d) Bulb decoctions administered to pshychotic patients(e) Bulb extracts taken orally by Sotho, Xhosa and Zulu for stress-related ailments(f) Bulb infusions drunk to relieve various mental illnesses, including anxiety and

depression(g) Bulb extracts used for age-related dementia

Verzár and Petri (1987)Watt and Breyer-Brandwijk (1962)Nielsen et al. (2004), Watt and Breyer-Brandwijk (1962)Nyazema (1986), Pedersen et al. (2008), Sobiecki (2008)Hutchings and Van Staden (1994)Neergaard et al. (2009), Pedersen et al. (2008), Sandageret al. (2005), Stafford et al. (2008)Risa et al. (2004a, 2011b), Stafford et al. (2008)

10. Inflammatoryconditions

(a) Milk in which bulb scales have been macerated was used by Europeans in theLydenburg district to treat various inflammatory conditions

(b) Moistened bulb scales were also known to be used by early settlers forrheumatic pain

(c) Bulbs are applied for eye conditions as well as healing of infected scars(d) Weak bulb decoctions administered orally or via enema to Zulu adults for

stomache, headache, chest and bladder pain(e) The Manyika apply bulb scales locally for relief from urticaria(f) Dried leaves moistened with milk or oil is used to treat skin diseases, varicose

ulcers and phlebitis(g) Fresh leaves applied by Zulu to check bleeding

Hutchings et al. (1996), Watt and Breyer-Brandwijk (1962)Botha et al. (2005), Van Wyk et al. (2002, 2005), Watt andBreyer-Brandwijk (1962)Verzár and Petri (1987), Watt and Breyer-Brandwijk (1962)Hutchings et al. (1996), Viladomat et al. (1997)Watt and Breyer-Brandwijk (1962)Mabona and Van Vuuren (2013), Watt and Breyer-Brandwijk (1962)Hutchings et al. (1996), Viladomat et al. (1997), Watt andBreyer-Brandwijk (1962)

J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 17

In addition to the widespread cultural significance of Boophonedisticha in areas as diverse as ‘arrow and dart poisons’ as well as‘initiation and circumcision rituals’, the plant is most widelyknown for several of its neurological functions such as treatmentof hysteria, stress-related ailments, psychosis, anxiety and depres-sion as well as age-related dementia (Watt and Breyer-Brandwijk,1962; Hutchings et al., 1996; Pedersen et al., 2008; Stafford et al.,2008) (Table 1). Interestingly, a global systematic survey of 143plants used to treat diseases of the nervous system showed thatseven representatives were from the family Amaryllidaceae,including Ammocharis coranica, Boophone disticha, Scadoxus puni-ceus, Galanthus nivalis, Lycoris radiata, Narcissus jonquilla andNarcissus pseeudonarcissus, of which the first three are African inorigin (Gomes et al., 2009). Furthermore, based on these findingsall plants covered in the survey were suggested as being possiblecomplementary or alternative therapies for neurological andneurodegenerative disorders (Gomes et al., 2009). An often over-looked area in this sector is the psychoactive properties ofBoophone disticha leading to it being commonly classified as an‘African hallucinogen’ (De Smet, 1996; Sobiecki, 2008; Foden2010). In the practice of trance, the Khoi-San believed that theplant could serve as a doorway to the spirit of the dead. Sangomasare also known to take the plant to psychoanalyse their patients, aprocess they call ‘bioscope’ hence the common name for Boophonedisticha (‘bioscope plant’) (De Smet, 1996; Sobiecki, 2008; Foden2010).

6. Phytochemistry

6.1. Alkaloids of the South African Amaryllidaceae

Although several classes of secondary metabolites are knownto occur in the Amaryllidaceae, such as chalcones, flavonoids,lectins, lignans, peptides and terpenoids, it is the isoquinolinealkaloids which have endowed the family with a unique chemical

characteristic (Viladomat et al., 1997; Bastida et al., 2006; Nairet al., 2013). These now collectively represent a large and expand-ing group of alkaloids with a distinct array of structural diversityin which galanthamine 1, lycorine 2 and crinine 3 feature as thethree major structural-types (Scheme 1) (Viladomat et al., 1997;Bastida et al., 2006; Jin, 2011; Nair et al., 2013). By contrast,homolycorine 4, tazettine 5 and montanine 6 make up the minorseries of compounds while trisphaeridine 7 and ismine 8 repre-sent some of the less-conspicuous members (Viladomat et al.,1997; Bastida et al., 2006; Jin, 2011; Nair et al., 2013). All thesestructures share the same biosynthetic precursor (norbelladine 9)from which they are derived via different sequences of phenolicoxidative coupling (Viladomat et al., 1997; Bastida et al., 2006; Jin,2011; Nair et al., 2013). To date over 500 of these alkaloids havebeen identified in Amaryllid species across the globe (Viladomatet al., 1997; Bastida et al., 2006; Jin, 2011; Nair et al., 2013). TheSouth African contingent presently stands at 193 alkaloids, repre-senting roughly 39% of the global complement of structures,which have been isolated from 88 species spanning 14 generaand three tribes (Viladomat et al., 1997; Nair et al., 2013).Furthermore, this number is dividable into the different groupsof compounds characteristic of the Amaryllidaceae, including 84crinanes, 40 lycoranes, 7 galanthamine compounds, 9 tazettinerepresentatives, 27 homolycorine compounds, 10 montanine ana-logues and 16 miscellaneous alkaloids (Viladomat et al., 1997; Nairet al., 2013). Although crinane alkaloids (such as crinine 3) with 84representatives are the most populous of the South AfricanAmaryllidaceae, lycorine 2 is by far the most common compoundhaving been isolated on 38 separate occasions i.e. it is present in43% of plants examined (Viladomat et al., 1997; Nair et al., 2013).Given this statistic alone coupled to the significant array ofbiological properties demonstrated by lycorine, it is not surprisingthat the Amaryllidaceae continues to attract widespread interestwith the status as a biologically privileged plant family. Further-more, the commercial success of galanthamine 1, the clinicalpotential of compounds such as pancratistatin 10 as well as the

Table 1 (continued )

Category of use Description of traditional usage References

11. Cancer Bulb extract indicated for this purpose Botha et al. (2005)

12. Malaria Bulb extract known to be effective in this treatment Watt and Breyer-Brandwijk (1962)

13. Tuberculosis Zulu in the Umtentweni area of KwaZulu-Natal are known to use bulb decoctions totreat tuberculosis

Watt and Breyer-Brandwijk (1962)

14. Psychoactiveproperties

(a) Weak bulb dococtions used as a sedative to calm psychotic patients(b) Bulb concoctions taken orally have caused sedation, analgesia, visual

hallucinations, unconsciousness, irrational behaviour, talkativeness as well ascoma

(c) Induce hallucinations for divinatory purposes(d) Used as a narcotic substance(e) Bulbs known to induce trance

Pedersen et al. (2008), Sobiecki (2008)Du Plooy et al. (2001), Neergaard et al. (2009)Van Wyk et al. (2002)Hutchings et al. (1996), Sandager et al. (2005)Neergaard et al. (2009), Stafford et al. (2008)

15. Divinatorypurposes

(a) The Manyika grow the plant outside their huts as a charm to ward off evildreams, to bring goodluck and rain as well as to keep away the ‘mudzimu’(wandering spirit) after a death

(b) Psychoactive properties of the bulbs used to induce trance(c) Used in various divination rituals such as arousing the ancestral spirits and

curing demonic possession(d) Thought to symbolise eternal life

Watt and Breyer-Brandwijk (1962)Neergaard et al. (2009), Stafford et al. (2008),Du Plooy et al. (2001), Gadaga et al. (2010), Hutchings et al.(1996), Philander (2011), Van Wyk et al. (2002)Van Wyk (2008)

16. Veterinary uses (a) The Xhosa use the bulbs as a remedy for redwater in cattle(b) Cattle are known to browse the plant with impunity(c) It has been suggested that vultures and other carrion birds eat the plant to

prevent harm from ingestion of putrid flesh or to sharpen vision

Watt and Breyer-Brandwijk (1962)Verzár and Petri (1987)Watt and Breyer-Brandwijk (1962)

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natural abundance of the Amaryllidaceae in South Africa, whereless than a third of the species have been studied, have togethergalvanised the plant family as a truly viable vehicle for drugdiscovery.

6.2. Alkaloids from Boophone disticha

Although various compounds such as furfuraldehyde, acetova-nillone, chelidonic acid, copper, laevulose, pentatriacontane, aphytosterol, ipuranol and fatty acids were initially reported to bepresent in bulbs of Boophone disticha (Tutin, 1911a), its alkaloidconstituents have subsequently gained prominence due to theirimportant biological properties. Given the long history of usage ofBoophone disticha in TM in South Africa, it is not surprising that theplant was amongst the first of the Amaryllidaceae species globallyto be examined for alkaloid constituents (Tutin, 1911a,b; Lewin,1912a,b; Tutin, 1913). Furthermore, the plant serves as a flagshipfor phytochemical endeavours emanating out of the southernAfrican region. Given the complexity of these structures, the earlyyears of chemical studies on Boophone disticha was understand-ably arduous. The first phytochemical investigation of the plantwas that performed by Tutin (1911a,b) who described the presenceof lycorine 2 together with three other unknown alkaloids.Following this Lewin (1912a,b) isolated a compound looselyreferred to as “haemanthine”, which Tutin (1913) maintainedwas in fact a mixture. However, evidence in support of Lewin'sstance for the chemical individuality of haemanthine was providedby Cooke and Warren (1953) based on elemental analysis, meltingpoints and semi-synthetic derivatisation. By contrast, support forthe stance taken by Tutin is reflected in studies carried out severaldecades later which revealed that “haemanthine” was a compo-nent mixture of buphanamine 11 or nerbowdine 12 (Bates et al.,1957; Goosen and Warren, 1957; Fales and Wildman, 1961).Following on these early investigations, Bates et al. (1957) isolatedlycorine 2, buphanamine 11 and buphanidrine 13 from bulbs ofBoophone disticha collected at various locations in South Africa,including Bloemfontein, Grahamstown, Pietermaritzburg and Pre-toria. However, to date the most comprehensive phytochemicalstudy carried out on the plant is that of Hauth and Stauffacher(1961) who described the presence of 11 alkaloids in ethanolicbulb extracts, including buphanidrine 13 (19.4%), undulatine 14(18.6%), buphanisine 15 (16.9%), buphanamine 11 (14.1%), nerbow-dine 12 (11.1%), crinine 3 (7.2%), distichamine 16 (5.4%), crinami-dine 17 (1.2%), acetylnerbowdine 18 (0.6%), lycorine 2 (0.4%) andbuphacetine (0.3%) (where percentage values are expressed asrelative contribution to total alkaloids). Whereas majority of thesecompounds have been subsequently verified through physical andspectroscopic means (Viladomat et al., 1997; Nair et al., 2013), theidentity of buphacetine remains a mystery to this day. Interest-ingly, buphanidrine, buphanisine, crinine and distichamine werealso found in Boophone haemanthoides, but at percentages of46.9%, 23.9%, 7.3% and 21.9%, respectively (Nair et al., 2012b).Further studies of Boophone disticha confirmed the presence ofbuphanamine, buphanidrine, buphanisine, crinine and disticha-mine (Sandager et al., 2005; Neergaard et al., 2009; Cheesmanet al., 2012). The most recent phytochemical investigation ofBoophone disticha led to the isolation of 6-hydroxycrinamine 19from a methanolic bulb extract at a concentration of 0.01% dryweight (Adewusi et al., 2012). It is clear from these collectivefindings that, with the exception of lycorine 2, the alkaloidsproduced by Boophone disticha are all the crinane series ofAmaryllidaceae alkaloids. Furthermore, 6-hydroxycrinamine 19was the only compound identified belonging to the α-crinanesub-series (Adewusi et al., 2012), the others being characteristic ofβ-crinane alkaloids (Hauth and Stauffacher, 1961; Sandager et al.,2005; Neergaard et al., 2009; Cheesman et al., 2012). In terms of

the structural features of these alkaloids, crinane compoundspossess a basic phenanthridine nucleus with varying degrees ofoxidation in ring-A, but usually with a methylenedioxy bridgeacross C-8 and C-9 (as in crinine 3 and buphanisine 15) (Viladomatet al., 1997; Bastida et al., 2006; Nair et al., 2013). In addition,methoxy substitution at C-7 is common for many analogues acrossthe series, such as buphanidrine 13 and undulatine 14 (Viladomatet al., 1997; Bastida et al., 2006; Nair et al., 2013). Substitution inring-B is infrequent, but when it is present it is normally at C-6 (asin 6-hydroxycrinamine 19) or C-4a (as in crinamabine 20)(Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013). Inaddition, protonated salts bearing chloride counter anions, as wellas N-oxide derivatives of several crinane compounds have beenidentified (Viladomat et al., 1997; Bastida et al., 2006; Nair et al.,2013). In relation to ring-C, oxygen-related substituents are knownto occur at all exchangeable positions, including C-1, C-2, C-3 andC-4, and a C-1 to C-2 double bond is common for many derivatives(Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013).A distinguishing feature of crinane alkaloids is the presence of theethano-bridge straddling N-5 and C-10b which may be either α- orβ-orientated, leading to the absolute stereochemical configurationat C-4a. In the case of lycorine 2, the C-terminus of the ethano-bridge is at C-4 and C-10b reverts to a tertiary carbon centre(Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013). Ofthe compounds isolated from Boophone disticha, distichamine 16 ismost noteworthy for its unique ring-C substitution pattern(Viladomat et al., 1997; Nair et al., 2012a, 2013). In its structure,oxidation has occurred at C-1 in accommodating the keto groupand the double bond has shifted to the C-2/C-3 position withconcomitant vinylization of the C-3 methoxy group (Viladomatet al., 1997; Nair et al., 2012b, 2013). Such a substitution patternhas never been found for any other alkaloid within the crinaneseries (Viladomat et al., 1997; Nair et al., 2012b, 2013). Further-more, distichamine 16 has never been found outside Boophone andthus reserves the privilege as a distinctive chemotaxonomicmarker for the genus (Viladomat et al., 1997; Nair et al., 2012b,2013).

7. Pharmacology

7.1. Antimicrobial activity

Given that Boophone disticha is the most common representa-tive of the South African Amaryllidaceae used to treat wounds andinfections (Hutchings et al., 1996; Rabe and Van Staden, 1997;Grierson and Afolayan, 1999; Shale et al., 1999), it is not surprisingthat some studies have been directed towards gauging its efficacyagainst bacterial pathogenesis (Heyman et al., 2009; Cheesmanet al., 2012). An ethanolic bulb extract of Boophone disticha wasactive against methicillin-sensitive Staphylococcus aureus with anMIC of 7.25 mg/mL (Heyman et al., 2009). Interestingly, the sameextract was shown to be potentially fatal to the pathogen as aminimum bactericidal concentration (MBC) was established at12.5 mg/mL (Heyman et al., 2009). Further work on Boophonedisticha by Cheesman et al. (2012) initially showed bulb extracts tobe active against two Gram-positive (Bacillus subtilis and Staphy-lococcus aureus) and two Gram-negative (Escherichia coli andKlebsiella pneumoniae) strains that made up the microdilutionbacterial screen, with IC50s in the range 0.13–0.25 mg/mL. Led bythese promising activities, bioassay-guided fractionation thenresulted in the isolation of two active crinane alkaloid constitu-ents: buphanidrine 13 and distichamine 16 (Scheme 1) (Cheesmanet al., 2012). In the repeat screen, best activities were seen againstStaphylococcus aureus, Escherichia coli and Klebsiella pneumoniaewith both compounds exhibiting IC50s of 0.063 mg/mL against all

J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 19

three pathogens (correspondent with micromolar values of 200and 191.5 for buphanidrine 13 and distichamine 16, respectively)(Cheesman et al., 2012). Both compounds were two-fold less activeagainst Bacillus subtilis (IC50 0.13 mg/mL) as also seen in the screenagainst the crude extract (Cheesman et al., 2012). These results,though not as potent as the neomycin standard (IC50s of 0.8�10–3–1.6�10–3 μg/mL) (Cheesman et al., 2012), are neverthelessconsistent with previous observations on the low-to-mild anti-bacterial activities of Amaryllidaceae alkaloids (Viladomat et al.,1997; Elgorashi et al., 2003; Bastida et al., 2006; Nair et al., 2013).Amongst the South African Amaryllidaceae only Boophone distichais known in TM for treating tuberculosis (Watt and Breyer-Brandwijk, 1962), although no pharmacological studies have beencarried out to verify this property.

Information pertaining to the usage of South African Amarylli-daceae plants for fungal infections is sparse in the literature.Although, Boophone disticha is not indicated for such purposes,several others, including Amaryllis belladonna, Crinum macowaniiand Crinum moorei, have shown antifungal or antiyeast activity(Viladomat et al., 1997). In terms of activities of single compoundisolates, lycorine 2 and vittatine 21 were reported with significantactivity against Candida albicans (MICs of 39 and 31 μg/mL,respectively) (Evidente et al., 2004). In relation to antiviral activity,again the literature is devoid of any mention of the traditionalusage of Boophone disticha for such purposes. However, the planthas been suggested to be effective against different rhinovirusstrains (Viladomat et al., 1997). In regards to activities of individualcompounds, lycorine 2 has been shown to be active against anumber of viruses (Bastida et al., 2006), including poliomyelitisvirus at concentrations as low as 1 μg/mL (Ieven et al., 1982).Structure–activity relationship studies involving Herpes simplexvirus showed that lycorine exerted its antiviral effect by blockingDNA polymerase activity (Ieven et al., 1983). Other compounds ofthe family which have demonstrated antiviral activity in modelstudies include homolycorine 4, tazettine 5, narciclasine 22 andhaemanthamine 23 (Scheme 1) (Bastida et al., 2006). In terms ofantiparasitic activity of South African Amaryllidaceae plants, onlyAmaryllis belladonna is known to be active against Plasmodiumgallinaceum (Viladomat et al., 1997). Amongst all these plants, onlyBoophone disticha is known to be used traditionally to treatmalaria (Watt and Breyer-Brandwijk, 1962), although no studieshave been directed towards gauging the efficacy of plant extractsagainst the malarial parasite. Of the various groups of Amarylli-daceae alkaloids screened against the malarial parasite, lycorine 2was the most potent against both chloroquine-sensitive (D10) andchloroquine-resistant (FAC8) strains of Plasmodium falciparumwith IC50s of 0.6 and 0.7 μg/mL, respectively (Campbell et al.1998, 2000; Şener et al., 2003).

7.2. Anti-inflammatory activity

‘Inflammation-related conditions’, ‘wounds and infections’ and‘ailments pertaining to the CNS’ together represent the top threecategories under which Boophone disticha finds most extensive use inTM in South Africa (Watt and Breyer-Brandwijk, 1962; Verzár andPetri, 1987; De Smet, 1996; Hutchings et al., 1996; Viladomat et al.,1997; Grierson and Afolayan, 1999; Du Plooy et al., 2001; Van Wyket al., 2008; Philander, 2011). Verification of the usage of the plant forinflammation-related conditions came from cyclo-oxygenase (COX)inhibition studies in which ethanolic bulb extracts of Boophonedisticha exhibited 55% inhibition against COX-1 (Jäger et al., 1996).In terms of the activities of single compound extractives, lycorine 2 isknown to possess anti-inflammatory properties as shown throughthe carrageenan-induced paw oedema test in mice (Citoglu et al.,1998). At a dosage of 100 mg/kg lycorine induced 20.1% inhibition ofoedema in the test subjects compared to 29.1% by the indomethacin

control (10 mg/kg) (Citoglu et al., 1998). In addition, crinine 3 andcrinamidine 17, both known alkaloid constituents of Boophonedisticha (Hauth and Stauffacher, 1961), exhibited mild anti-inflammatory activity as determined through COX inhibition(Elgorashi et al., 2003). To this extent, crinine 3 and crinamidine 17at a concentration of 500 μM exhibited respective inhibitory activitiesof 16% and 10% against COX-1 (Elgorashi et al., 2003). Interestingly,this activity was seen to be selective as COX-2 remained unaffectedby similar treatments using either compound (Elgorashi et al., 2003).

7.3. Effects on the central nervous system

7.3.1. Hallucinogenic effectsThe use of plants as a source of chemicals with CNS-activating

properties is common on the South African cultural landscape. Assuch, over 300 plant taxa have been identified within this contextof TM (Stafford et al., 2008). At least three Amaryllid plants areknown from local tradition to be exploited for hallucinatorypurposes (De Smet, 1996; Viladomat et al., 1997; Stafford et al.,2008). Pancratium tenuifolium is used by the San of the Kalahari toinduce visual hallucinations while Brunsvigia radulosa is well-known from San culture as a psychoactive plant (Viladomatet al., 1997). In addition, Boophone disticha is recognised as ahallucinogenic plant in both San and Sotho traditions (Viladomatet al., 1997; De Smet, 1996; Neuwinger, 1994; Neuwinger andMebs, 1997; Sobiecki, 2002, 2008; Stafford et al., 2008). It has beensuggested that its alkaloid constituents, either as single com-pounds or additively, are largely responsible for these effects ofthe Amaryllidaceae (Viladomat et al., 1997; Bastida et al., 2006;Nair et al., 2013). Of the Amaryllidaceae alkaloids, galanthamine 1(Scheme 1) is best known for its narcotic effects such as inductionof “lucid dream” (LD) and “out of body experience” (OBE) as wellas its application as a nootropic agent (Harvey, 1995; Heinrich andTeoh, 2004). Of the compounds isolated from Boophone disticha,buphanidrine 13 is known to have hyoscine-like activity i.e. itexhibits muscarinic antagonist effects (Viladomat et al., 1997; Nairet al., 2013). Furthermore, since the identity of buphacetine hasnever been clarified, it could be speculated that this alkaloid maybe responsible for the hallucinogenic properties of the plant. Inaddition, it is possible that all (or some) the alkaloids known fromBoophone disticha could together function in a synergistic mannerto produce the observed hallucinogenic effects. Symptoms of theseeffects include unconsciousness, dilated pupils, tachycardia, raisedblood pressure, slightly raised temperature, laboured respiration,psychosis, drunkenness and visual disturbances as reported byLaing (1979) subsequent to treating patients presenting withBoophone disticha intoxication.

7.3.2. Mental disorders7.3.2.1. Anxiety and stress. In addition to the psychoactive propertiesof Boophone disticha, the plant is also used to treat mental disorderssuch as anxiety, depression, epilepsy and age-related dementia(Stafford et al., 2008; Gomes et al., 2009). Anxiety disorders are themost common psychiatric illnesses encountered in clinical practice(Pote et al., 2013). Often they are chronic conditions associatedwith considerable cardiovascular-related morbidity and mortality(Schwartz and Nihalani, 2006). Although information pertaining tothe pharmacological basis to the usage of Boophone disticha foranxiety and stress is largely absent from the literature, a recent studyemployed a mouse model to validate this claim (Pote et al., 2013).Maternal separation is a common animal model used to examine thelong-term effects of early life experience on subsequent behaviour inadulthood and the development of psychiatric disorders such asanxiety and depression (Pryce and Feldon, 2003). Furthermore,repeated early maternal separation stress is known to produce an

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altered effect in cardiovascular response in such models of study(Pryce and Feldon, 2003). In this regard, ethanolic extracts ofBoophone disticha were used to gauge the effects on blood pressureand heart rate in adult BALB/c mice subjected to repeated earlymaternal separation stress (Pote et al., 2013). The results revealedthat maternally separated mice treated with a low dose (10 mg/kg/BW/day) of Boophone disticha had the lowest systolic blood pressure,whereas those treated with a high dose (40 mg/kg/BW/day) exhibitedthe lowest diastolic blood pressure and mean arterial pressurefollowing acute stress in adulthood compared to the control anddiazepam treated animals (Pote et al., 2013). These findings suggestthat postnatal stress can induce short-term changes in the sensitivityof the cardiovascular system to subsequent stress which can bereduced by treatment with Boophone disticha.

7.3.2.2. Depression. Several antidepressant drugs available in clinicalpractice, such as citalopram, fluoxetine and paroxetine, exert theireffects through selective inhibition of serotonin reuptake and arepharmaceutically classed as selective serotonin reuptake inhibitors(SSRIs) (Nielsen et al., 2004). Mechanistically, such compounds areknown to bind to the SSRI binding site on the neuronal serotonintransporter thus inhibiting the transport of serotonin from thesynaptic gap back to the neuron (Stahl, 1998). In the competitivebinding assay with 3H-labelled citalopram, aqueous leaf and bulbextracts of Boophone disticha resulted in the displacement of morethan 50% of the transport protein bound [3H]citalopram at testedconcentrations of 5, 1 and 0.1 mg/mL (Nielsen et al., 2004). Buoyed bythese findings, bioassay-guided fractionation then led to the isolationof the alkaloids buphanamine 11, buphanidrine 13, buphanisine 15and distichamine 16 (Scheme 1) from bulbs of Boophone disticha,which exhibited respective IC50 values of 55, 62, 199 and 65 μM in theserotonin binding assay (Neergaard et al., 2009). In addition,buphanamine 11 and buphanidrine 13 demonstrated affinities to theserotonin transporter in rat brain with IC50s of 1799 and 274 μM,respectively (Sandager et al., 2005). Apart from this, Pedersen et al.(2008) employed a rat model for depression to show that Boophonedisticha extracts inhibited the serotonin transporter (SERT),noradrenalin transporter (NAT) as well as the dopamine transporter(DAT), all of which are believed to be involved in the pathophysiologyof depression, with IC50s of 423.8, 77.3 and 93.5 μM respectively.Furthermore, antidepressant-like effects were observed for extracts ofBoophone disticha in the tail suspension test (TST) and the forced swimtest (FST) in both rats and mice (Pedersen et al., 2008).

7.3.2.3. Epilepsy. Epilepsy is a neurological condition typified byrepeated and unprovoked seizures often accompanied by severeconvulsions (Risa et al., 2004a). Benzodiazepines are a common classof anti-convulsing agents which bind to the GABAA�benzodiazepinereceptor complex where they enhance the affinity for the inhibitoryneurotransmitter γ-aminobutyric acid (GABA) (Risa et al., 2004a).A GABA stimulus on the GABAA-receptor causes an influx of chlorideions into the cell producing hyperpolarisation of the membrane,making it more difficult to generate action potential, resulting in cellarrest and consequent anti-convulsant activity (Risa et al., 2004a).Although Boophone disticha is known in the TM approach to epilepsy(Stafford et al., 2008), no pharmacological investigation has sought toclarify this usage. By contrast, the Amaryllid Brunsvigia grandifloraexhibited high binding affinities (up to 100%) for the GABAA-benzodiazepine receptor in competition with flumazenil (Risa et al.,2004a). Furthermore, none of the components of a structurallydiverse Amaryllidaceae alkaloid mini-library were responsive toGABAA-benzodiazapine receptor binding (Elgorashi et al., 2006b).

7.3.2.4. Age-related dementia. The impact of age-related dementiaon western medicine is staggering, affecting one in 20 people over

65 and one in five over the age of 80, with an estimated annualcost of over $600 billion (McNulty et al., 2010; Nair and VanStaden, 2012). By contrast, CNS disorders associated with old agesuch as AD, amyotrophic lateral sclerosis (ALS) and Parkinson'sdisease (PD) are not a major concern for the healthcare sectors ofdeveloping nations, which has been attributed to a number offactors, including genetic, dietary, lifestyle and environmentalmeasures entrenched in these societies (Stafford et al., 2008).Given this rising health concern for developed nations, the searchand development of chemical targets into suitable therapiesagainst this complex of neuronal diseases is at a premium(Heinrich and Teoh, 2004; Houghton et al., 2006; Nair and VanStaden, 2012). In this regard, plants provide a substantial resourcebase for such efforts in drug discovery, some of which have alreadydelivered effective candidates such as rivastigmine, huperzine Aand galanthamine (also referred to as galantamine) to the clinicalmarket (Heinrich and Teoh, 2004; Houghton et al., 2006). Thesecompounds are highly efficient at AChE inhibition (IC50 forgalanthamine is 1 μM), a process thought to be significant in thepathophysiology of AD, which fits the premise that Alzheimer'sdisease is associated with cholinergic insufficiency (Harvey, 1995;Greig et al., 2004; Heinrich and Teoh, 2004; Houghton et al., 2006).

Given the commercial success of galanthamine 1 (Scheme 1), it isclear that the plant family Amaryllidaceae invokes widespreadinterest as a resourceful platform for discovery in this area (Lopezet al., 2002; Elgorashi et al., 2004, 2006a; McNulty et al., 2010;Monton et al., 2010; Nair et al., 2011; Nair and Van Staden, 2012).Such endeavours have highlighted the potency of other alkaloidssuch as sanguinine 24 (IC50 0.1 μM) and 1-O-acetyllycorine 25 (IC500.96 μM) (Scheme 1) as AChE inhibitors with genuine potential forclinical advancement (Lopez et al., 2002; Elgorashi et al., 2004,2006a; McNulty et al., 2010; Monton et al., 2010; Nair et al., 2011;Nair and Van Staden, 2012). The activity of sanguinine is notunexpected given its close structural similarity to the standard AChEinhibitor galanthamine. However, its potency over galanthaminemust reside with its ring-A phenolic hydroxyl group (which in thecase of galanthamine is substituted by a methoxyl group). The role ofthe methoxyl group in galanthamine has been shown to be sig-nificant as it occupies the acyl-binding pocket, contacting both Phe-288 and Phe-290 amino acid residues within the active site, and istherefore an essential structural feature of this AChE inhibitorypharmacophore (Sussman et al., 1991; Greenblatt et al., 1999). Theactivity of sanguinine 24 over galanthamine 1 suggests that a smallpolar group in the C-9 region of the molecule, capable of hydrogenbond donor and acceptor functions (reminiscent of a phenolichydroxyl), is better accommodated at the active site.

The potency of 1-O-acetyllycorine 25 at AChE inhibition is duechiefly to its structural proximity to galanthamine and the possibilityof overlap along significant areas of their respective pharmacophores(Elgorashi et al., 2006a; Nair et al., 2011). Quantitative structure–activity relationship (QSAR) studies involving superposition of mini-mum energy conformations showed partial overlap for the methoxylof galanthamine and the methylenedioxy of 1-O-acetyllycorine(Elgorashi et al., 2006a; Nair et al., 2011). Furthermore, the acetateand nitrogen atom of 1-O-acetyllycorine superimposed on thehydroxyl group and the nitrogen atom of galanthamine, respectively(Elgorashi et al., 2006a; Nair et al., 2011), making it possible for theacetyl group to participate in hydrogen bonding in a manner similarto that demonstrated for the hydroxyl of galanthamine (Greenblattet al., 1999). In addition, the C-4/C-4a double bond in galanthaminewas seen to have π�π interactions with Trp-84 (Greenblatt et al.,1999), and since the lycorine double bond is in a proximally similarposition it may be able to elicit a similar response at the active site(Elgorashi et al., 2006a; Nair et al., 2011).

Boophone disticha is one of the seven different South AfricanAmaryllidaceae plants shown to be effective in the treatment of

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age-related dementia and memory loss in TM (Stafford et al.,2008). As such, verification of this property was arrived at viaAChE inhibition assays by which aqueous and ethanolic bulbextracts exhibited inhibitory activities of 37% and 30%, respectively(Risa et al., 2004b). Of the 12 compounds isolated from Boophonedisticha, lycorine 2, 6-hydroxycrinamine 19 and crinine 3 exhibitedAChE inhibitory activities of 29.3, 445 and 461 μM respectively(Elgorashi et al., 2004; Adewusi et al., 2012; Elisha et al., 2013),which consolidate the traditional usage of the plant for theseconditions (Stafford et al., 2008; Gomes et al., 2009).

In addition to AChE inhibitors, monoamine oxidase (MAO)inhibitors are also known to be effective in the treatment of ADand PD (Stafford et al., 2007). MAO enzymes are present in theouter mitochondrial membrane of neuronal and non-neuronalcells and are responsible for the oxidative deamination of endo-genous as well as xenobiotic amines (Yamada and Yasuhara, 2004).The two known isoforms MAO-A and MAO-B are differentiated bysubstrate preference, inhibitor specificity and tissue distribution(Yamada and Yasuhara, 2004). MAO-A preferentially deaminatesserotonin, adrenaline and noradrenaline and makes up around 75%of MAO in human brain tissue, while MAO-B is specific forsubstrates such as dopamine, β-phenylethylamine and benzyla-mine (Yamada and Yasuhara, 2004). In relation to this, Staffordet al. (2007) screened a collection of 20 different plant taxa knownto be used in South African TM for age-related dementia for MAOactivity. In the study including the two Amaryllids Boophonedisticha and Scadoxus puniceus, only the latter was active in non-selective MAO inhibition (IC50 406 μg/mL) and selective MAO-Binhibition (IC50 344 μg/mL) (Stafford et al., 2007). This resulthighlights the utility of Boophone disticha as a source of selectivechemotherapeutics against motor neuron disease, given its activityin AChE inhibition.

7.4. Anticancer activity

Cancer is a leading cause of death worldwide, accounting for7.6 million deaths (around 13% of all deaths) in 2008 (WHO, 2008).During this period 669,000 deaths as a result of cancer, including349,000 in men and 320,000 in women, were recorded in NorthAmerica (WHO, 2008). Such statistics for the African continent aremore difficult to come by since a very small proportion of the totalpopulation is covered by medically certified causes of death orpopulation-based cancer registries (WHO, 2008). However, con-servative estimates have put this number around 518,000 fordeaths from cancer in the African region for 2008 (WHO, 2008).Of greater concern is that global deaths from cancer are set to risesharply with 17 million cases projected for 2030 (WHO, 2008). InNorth America, lung, stomach, liver, colon and breast cancerstogether cause the most number of deaths from cancer each year,of which lung cancer is forecast as the leading cause for bothmales and females in 2013 (Cancer Facts & Figures, 2013). Giventhese facts and their increasing burden on the healthcare sector,the search for effective new therapies is an ongoing concern forhealth organisations worldwide. Present therapies include che-motherapy, immunotherapy, monoclonal antibody therapy, radia-tion therapy and surgery. In this regard, plants provide a vastresource base for chemotherapeutic based anti-cancer drug dis-covery (Graham et al., 2000; Balunas and Kinghorn, 2005; Craggand Newman, 2005; Pan et al., 2010). As such, over 60% of anti-cancer agents presently in clinical use are derived from naturalsources, including plants, marine organisms and micro-organisms(Cragg and Newman, 2005; Pan et al., 2010). Examples of suchcommercial drugs originating in plants include vinblastine, vin-cristine, camptothecin, topotecan, irinotecan, etoposide and pacli-taxel (Cragg and Newman, 2005; Pan et al., 2010). In the SouthAfrican context, the work of Fouche et al. (2006, 2008) is most

embracive of the anti-cancer properties of the country's floralbiodiversity. To this extent, 7500 randomly selected plant extracts(representing 700 species) were tested in the three cell line pre-screen using MCF7, TK10 and UACC62 cells at a single dose of100 μg/mL (Fouche et al., 2006, 2008). Out of these, a total of 950extracts exhibited growth inhibitions of 75% or higher against twoor more of the cancer cells screened (Fouche et al., 2006, 2008).

The traditional use of Amaryllidaceae plants for cancer isknown from all three geographical regions of its prominence,including Andean South America, the Mediterranean and SouthAfrica (Pettit et al., 1984; Nair et al., 1998; Botha et al., 2005;Kornienko and Evidente, 2008; Caamal-Fuentes et al., 2011).Amaryllis belladonna, Boophone disticha and Crinum delagoenseare three of the local Amaryllid plants known to be used in TMfor treating this disease (Botha et al., 2005; Nair et al., 1998; Pettitet al., 1984). Apart from its clinical relevance to MND, the plantfamily Amaryllidaceae is most widely known in pharmacology forits anti-cancer properties (Kornienko and Evidente, 2008;Evidente and Kornienko, 2009; Nair et al., 2012a). Furthermore,its alkaloid constituents are known to be responsible for theseeffects as shown via both in vitro and in vivo models of study(Kornienko and Evidente, 2008; Evidente and Kornienko, 2009;Nair et al., 2012a). Although cytotoxic activities are detectableacross all series of alkaloids of the Amaryllidaceae, the crinane andlycorine alkaloids are the most common targets of choice in suchstudies due to their potency (Evidente and Kornienko, 2009;Lamoral-Theys et al., 2010; Nair et al., 2012a). As such, over 100different crinane and lycorine structures have been evaluated foranti-tumour activity (Evidente and Kornienko, 2009; Lamoral-Theys et al., 2010; Nair et al., 2012a). Of the 12 alkaloids reportedfrom Boophone disticha (Hauth and Stauffacher, 1961; Sandageret al., 2005; Neergaard et al., 2009; Adewusi et al., 2012;Cheesman et al., 2012), all with the exception of nerbowdine 12,acetylnerbowdine 18 and buphacetine have been subjected tocytotoxicity screening against various cancer cells (Evidente andKornienko, 2009; Lamoral-Theys et al., 2010; Nair et al., 2012a). Inthis regard, studies on lycorine 2 (Scheme 1) are significant in thatit was amongst the first of the Amaryllidaceae alkaloids to exhibitcytotoxic activity as shown by its inhibitory effects towards celldivision and cell elongation, as well as protein synthesis ineukaryotic cells (De Leo et al., 1973; Jimenez et al., 1976).Furthermore, both in vitro and in vivo models of leukaemia (HL-60) cells support the potency of lycorine as an antiproliferativeagent (Liu et al., 2004, 2007). Notably, in some cell lines theseeffects were manifested via the apoptotic mechanistic pathway(Liu et al., 2007; Li et al., 2007; McNulty et al., 2009a). By contrast,antiproliferative effects by lycorine 2 were also exerted inapoptosis-resistant cell lines such as OE21 oesophageal cancerand SKMEL-28 melanoma cells (Van Goietsenoven et al., 2010). Interms of potency, IC50s as low as 0.5 μMwere observed for lycorine2 against various leukaemia and lymphoma cell lines, while forcarcinomas, multiple myeloma and melanoma cells IC50s were inthe range 3–10 μM (Lamoral-Theys et al., 2010). In vivo, HL-60leukaemia-bearing mice exhibited a T/C index of 134% at a lycorinetreatment dosage of 10 mg/kg (Lamoral-Theys et al., 2010). Simi-larly, crinane alkaloids of the Amaryllidaceae have come into focussince the identification of haemanthamine 23 as a cytotoxic agentover 30 years ago (Jimenez et al., 1976). Of the crinanes identifiedin Boophone disticha, buphanisine 15 has been most studied,having been screened against 22 different cancers with the bestactivity seen in Vinblastine-resistant oral epidermoid KB-V1 carci-noma cells [ED50 19.8 μg/mL (69.5 μM)] (Likhitwitayawuid et al.,1993; Nair et al., 2012a). 6-Hydroxycrinamine 19, the only α-crinane of the compounds isolated, exhibited strong activityagainst mouse embryonic fibroblasts (3T3) (MTD 0.63 μM) andmoderate activity against human neuroblastoma (SH-SY5Y) cells

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(IC50 54.5 μM) (Adewusi et al., 2012; Nair et al., 2012a). Activitiesfor buphanidrine 13, buphanamine 11, crinamidine 17, crinine 3and undulatine 14 were generally moderate to mild with crinineshown to be most effective against the multi-drug resistant humanmyeloid leukaemia cell line HL-60/Dox (IC50 14.04 μM) (Berkovet al., 2011; Nair et al., 2012a). Finally, out of a mini-panel of fivecancer cells, distichamine 16 exhibited best activity against HeLacervical adenoacarcinoma (IC50 2.2 μM) (Nair et al., 2012a,c).Information pertaining to in vivo studies of crinane alkaloids isgenerally lacking in the literature; however, one study describedsignificant reductions in the growth of sarcoma 180 ascitestumours in mice after treatment with various β-crinanes (Ghosal,1986; Nair et al., 2012a). In relation to the mechanistic basis tothese alkaloids, the apoptotic mode of death in cancers has beendemonstrated for crinine 3, distichamine 16, crinamine 26 andhaemanthamine 23, the first two of which are significant as theyare constituents of Boophone disticha (Griffin et al., 2007; McNultyet al., 2007; Van Goietsenoven et al., 2010; Berkov et al., 2011; Nairet al., 2012c). For example, McNulty et al. (2007) showed that up to90% of rat hepatoma (5123 tc) cells exhibited apoptotic morphol-ogy after a 48 h treatment with either crinamine or haemanth-maine (ED50s 12.5 and 15 μM, respectively). Interestingly, thisactivity was seen to be selective as normal human embryonickidney (293t) cells remained unaffected by the treatment (Griffinet al., 2007; McNulty et al., 2007; Nair et al., 2012a). Apart fromthis Berkov et al. (2011) demonstrated via oligonucleosomal DNAfragmentation that the cytotoxicity of crinine 3 in HL-60/Dox cells(IC50 14.04 μM) ensues via the apoptosis pathway. In addition,caspase-mediated apoptosis induction has recently been describedfor distichamine 16 in lymphoblastic leukaemia (CEM) cells (IC50

4.5 μM) (Nair et al., 2012a,c). As such, flow cytometric analysisshowed that treatment with distichamine 16 increased the pro-portion of G2/M phase cells in a dose-dependent manner, withconcomitant reductions in the proportion of G0/G1 and S cells(Nair et al., 2012a,c). In addition, the proportion of cells with sub-G1 amounts of DNA (apoptotic cells) increased (up to 23.7%)following a 24 h treatment with distichamine indicating that thecompound was capable of cell cycle disturbance and apoptosisinduction (Nair et al., 2012a,c). Furthermore, distichamine induceda 12.5-fold increase in caspase-3/7 activity after 24 h at the highesttested concentration (20 μM) compared to untreated controls (Nairet al., 2012a,c). In addition to this, several changes in the expres-sion of apoptosis-related proteins were detected via Westernblotting in CEM cells following treatment with distichamine(Nair et al., 2012a,c). For example, after 24 h at 10 and 20 mMcleavage of PARP (89 kDa fragment) was observed which alsocorresponded with decreased levels of procaspase-3 (Nair et al.,2012a,c). Alongside this, expression of the tumour suppressorprotein p53 was discernible in the CEM cell control, and disticha-mine caused its enhanced expression after 24 h, notably at 10 and20 mM (Nair et al., 2012a,c). No change in the expression of theantiapoptotic protein Bcl-2 was observed but at 20 mM a decreasedlevel of the anti-apoptotic protein Mcl-1 was detected indicatingthe onset of apoptosis (Nair et al., 2012a,c). The traditional usage ofBoophone disticha in cancer therapy can therefore be ratifiedthrough several sources of convincing pharmacological evidence.

7.5. Toxicological studies

Despite the manifold benefits associated with the usage ofBoophone disticha in TM, the plant is nevertheless one of severalSouth African Amaryllids classified as poisonous (Van Wyk et al.,2002, 2005). This is understandable given the long cultural historyof Boophone disticha as an arrow poison (Watt and Breyer-Brandwijk, 1962; Bisset, 1989; De Smet, 1998; Hutchings et al.,1996).

Interestingly, forensic examination of an arrow taken from aSan community in the Cape (Section 5) and known to have beentreated with Boophone disticha showed that the arrow poison wascapable of killing mice within 20–30 min in subcutaneous doses of100–300 μg (De Smet, 1998; Mebs et al., 1996). Although animalsand birds are known to consume various parts of the plant(Table 1, entry 16), such tendencies have not been observed inhumans (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996).However, the widespread usage of bulb concoctions, decoctionsand infusions suggest that these are obviously not taken at lethaldosages (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996).In cases where poisoning with Boophone disticha were reported,the toxic effects produced included nausea, coma, muscularflaccidity, visual impairment, stertorous breathing, respiratoryparalysis, feeble or increased pulse, dyspnoea, and hyperaemiaand oedema of the lungs (Watt and Breyer-Brandwijk, 1962;Nyazema, 1986; Hutchings et al., 1996; Du Plooy et al., 2001).

Acute oral toxicity studies revealed that extracts of Boophonedisticha were lethal to Sprague Dawley rats at doses equal to orhigher than 240 mg/kg, killing animals between 30 min and 3 hafter dose administration, with an LD50 determined between 120and 240 mg/kg (Gadaga et al., 2010). Furthermore, in the samestudy the most prominent neurotoxicological effects linked todoses higher than 240 mg/kg were increased flaccid limb paralysis,retropulsion and hypoactivity which were evident from significantrearing, reduced mobility and gait scores (Gadaga et al., 2010). Inaddition, it was suggested that mydriasis, palpebral closure,tachypnoea, piloerection and spastic hind limb paralysis observedat lower doses of Boophone disticha could be due to the antic-holinergic effects of its alkaloid constituent lycorine 2 (Scheme 1)(De Smet, 1996; Gadaga et al., 2010). The sub-acute toxic effects ofBoophone disticha appear to be mediated via interference with theneuronal pathways especially the central dopaminergic and motorneurons (Gadaga et al., 2010). Histopathological observationssuggested the liver, small and large intestines, stomach, centralnervous system and peripheral nervous system as the targetorgans (Gadaga et al., 2010). A further investigation was carriedout to ascertain whether pre-treatment of Boophone distichapoisoned BALB/c mice with a CNS acting serotonin antagonist(cyproheptadine) had a protective effect from toxicity and mor-tality (Mutseura et al., 2013). Using the Functional ObservationalBattery (FOB) test to evaluate neurobehavioral and physiologicalchanges associated with toxicity, Mutseura et al. (2013) showedthat cyproheptadine pre-treatment (15–20 mg/kg) led to a dose-dependent decrease in mortality from 80% in untreated animals to30% in the pre-treated group, as well as a reduction in other toxicsymptoms typically associated with Boophone disticha poisoning(Gadaga et al., 2010).

Apart from this, based on the putative effects of Boophonedisticha on inflammatory response and immune function, extractsof the plant were also investigated for ATP production effects inisolated human neutrophils (Botha et al., 2005). To this extent,ethanolic bulb extracts were shown to significantly decrease ATPas well as superoxide production in the neutrophil medium (Bothaet al., 2005). A decrease in intracellular ATP is known to reflectpossible cell injury due to toxicity to neutrophils (Crouch et al.,1993). Conversely, it was suggested that the therapeutic benefits ofBoophone disticha may reside with its ability to inhibit superoxiderelease from neutrophils (Botha et al., 2005). Studies on thegenotoxic effects of Boophone disticha showed extracts of the plantto be genotoxic to human lymphocytic cells as determined by themicronucleus test (Taylor et al., 2003). For example, in the testagainst aqueous methanol bulb extracts the number of micro-nuclei produced per thousand binucleated cells in the lympho-cytes was 8, 14 and 2 at set concentrations of 100, 500 and2500 ppm, respectively, with a control value of 3 (Taylor et al.,

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2003). Furthermore, no extracts of Boophone disticha exhibitedanti-mutagenic effects as determined by the Ames test(Verschaeve and Van Staden, 2008). Of the alkaloids identified inBoophone disticha, lycorine 2 is known to exhibit toxic effects(Watt and Breyer-Brandwijk, 1962; Van Wyk et al., 2002, 2005).For example, in dogs it exhibited an LD50 of 41 mg/kg and thesymptoms of poisoning included salivation, vomiting, diarrhoeaand paralysis (Watt and Breyer-Brandwijk, 1962; Van Wyk et al.,2002, 2005). In addition, subcutaneously administered lycorineinduced nausea and emesis in beagle dogs starting at 0.5 mg/kgand reaching statistical significance at 1 mg/kg (Kretzing et al.,2011). These symptoms were short lasting and occurred no laterthan 2.5 h post-treatment (Kretzing et al., 2011). In the same studylycorine exhibited linear plasma kinetics with a mean eliminationhalf-life of 0.67 h after a single subcutaneous dose and 0.3 h afterintravenous administration (Kretzing et al., 2011). Although lycor-ine 2 is well-known within the Amaryllidaceae for its potentcytotoxic properties (Evidente and Kornienko, 2009; Lamoral-Theys et al., 2010), it has nonetheless shown to be non-selectiveat this activity. For example, in a screen involving the five humancancer cells: acute lymphoblastic leukaemia (CEM), chronic mye-logenous leukaemia (K562), breast adenocarcinoma (MCF7),malignant melanoma (G-361) and cervical adenocarcinoma(HeLa), IC50s determined against lycorine were 1.6, 3.6, 13.0, 10.6and 5.0 μM, respectively (Nair et al., 2012c). However, in the samestudy the compound also targeted normal human fibroblast (BJ)cells with an IC50 of 1.9 μM, thus highlighting a serious concern forsuch compounds in cancer chemotherapy (Nair et al., 2012c).

An often neglected area in TM is the interaction of plant-derived (or other) products with the cytochrome enzymes. Cyto-chromes P450 are heme-containing monoxygenase enzymes thatare expressed primarily on smooth ER membranes of hepatocytesand by cells along the intestinal tract mucosal surface (Guengerich,1997). They are involved in the detoxification of a wide variety ofxenobiotics such as drugs, biogenic amines from food sources,environmental toxins, and chemical carcinogens, and in theoxidation of steroids, fatty acids, prostaglandins, leukotrienes,and fat-soluble vitamins (Guengerich, 1997). The CYP3A subfamilycomprises about 30% of the total liver cytochrome P450 enzymepool in humans, and the isoenzyme CYP3A4 accounts for approxi-mately 60% of drugs metabolised (Guengerich, 1997). Althoughextracts of Boophone disticha have not been screened for CYP3A4activity, two of its alkaloid constituents (lycorine 2 and crinine 3)were inactive against the enzyme when measured against keto-conazole as standard (Ki 0.024 μM) (McNulty et al., 2009b). Bycontrast, 1-(O)-acetyllycorine 25 was moderately active with a Kivalue of 7.6 μM (McNulty et al., 2009b).

8. Conclusions

Boophone disticha is worthy of its standing as a flagshiprepresentative of the South African Amaryllidaceae. Such recogni-tion is based on its widespread examination in the three thematicareas highlighted as the focus of this review: traditional usage,phytochemistry and pharmacology. The traditional usage of theplant was shown to be quite broad, ranging from cultural anddietary applications to treatment of personal injury and terminalmedical conditions such as cancer. In addition, such usage wasseen to incorporate six body systemic functions. Three categorieswere identified in which the plant finds most extensive usage,including (i) ailments pertaining to the CNS; (ii) wounds andinfections; and (iii) inflammatory conditions. The chemical con-stituents of Boophone disticha were shown to primarily involveisoquinoline alkaloids which are unique to and characteristic ofthe plant family Amaryllidaceae. More importantly, several of

these entities have been shown to be chiefly responsible for thevarious biological effects exhibited by the plant, of which its use inthe traditional treatment of cancer is most striking. Despite this,several areas of its usage in TM still require pharmacologicalvalidation. Furthermore, stringent efforts are required to furtherdelineate the well-documented toxicological properties of Boo-phone disticha. As such, these collective efforts should go someway towards maximising the potential of Boophone disticha as abeacon for the plant family Amaryllidaceae in South Africantraditional medicine.

Acknowledgements

Financial assistance in support of this work by the University ofKwaZulu-Natal is gratefully acknowledged.

References

Adewusi, E.A., Fouche, G., Steenkamp, V., 2012. Cytotoxicity and acetylcholinester-ase inhibitory activity of an isolated crinine alkaloid from Boophone disticha(Amaryllidaceae). J. Ethnopharmacol. 143, 572–578.

Balunas, M.J., Kinghorn, A.D., 2005. Drug discovery from medicinal plants. Life Sci.78, 431–441.

Barnard, A., 1992. Hunters and Herders of Southern Africa: A ComparativeEthnography of the Khoisan Peoples. Cambridge University Press, Cambridge.

Bastida, J., Lavilla, R., Viladomat, F., 2006. Chemical and biological aspects ofNarcissus alkaloids. In: Cordell, G.A. (Ed.), The Alkaloids, vol. 63. Elsevier,Amsterdam, pp. 87–179.

Bates, A.N., Cooke, J.K., Dry, L.J., Goosen, A., Krüsi, H, Warren, F.L., 1957. Amar-yllidaceae alkaloids. II. Haemanthine and a new alkaloid distichine fromBoophone disticha Herb. J. Chem. Soc., 2537–2540.

Berkov, S., Romani, S., Herrera, M., Viladomat, F., Codina, C., Momekov, G., Ionkova,I., Bastida, J., 2011. Antiproliferative alkaloids from Crinum zeylanicum. Phyt-other. Res. 25, 1686–1692.

Binneman, J., 1999. Mummified human remains from the Kouga mountains, EasternCape. Digging Stick 16, 1–2.

Bisset, N.G., 1989. Arrow and dart poisons. J. Ethnopharmacol. 25, 1–41.Botha, E.W., Kahler, C.P., Du Plooy, W.J., Du Plooy, S.H., Mathibe, L., 2005. Effect of

Boophone disticha on human neutrophils. J. Ethnopharmacol. 96, 385–388.Brueton, V.J., Williams, V.L., Witkowski, E.T.F., 2008. Size class structure of three

commonly traded bulbs at Faraday (Johannesburg) umuthimarket: implicationsfor sustainability. S.Afr. J. Bot. 74, 362.

Caamal-Fuentes, E., Torres-Tapia, L.W., Simá-Polanco, P., Peraza-Sánchez, S.R., Moo-Puc, R., 2011. Screening of plants used in Mayan traditional medicine to treatcancer-like symptoms. J. Ethnopharmacol. 135, 719–724.

Campbell, W.E., Nair, J.J., Gammon, D.W., Bastida, J., Codina, C., Viladomat, F., Smith,P.J., Albrecht, C.F., 1998. Cytotoxic and antimalarial alkaloids from Brunsvigialittoralis. Planta Med. 64, 91–93.

Campbell, W.E., Nair, J.J., Gammon, D.W., Codina, C., Bastida, J., Viladomat, F., Smith,P.J., Albrecht, C.F., 2000. Bioactive alkaloids from Brunsvigia radulosa. Phyto-chemistry 53, 587–591.

Cancer Facts & Figures, 2013. American Cancer Society: Atlanta.Cheesman, L., 2013. Micropropagation and pharmacological evaluation of Boophone

disticha (Ph.D. dissertation). University of KwaZulu-Natal, South Africa.Cheesman, L., Nair, J.J., Van Staden, J., 2012. Antibacterial activity of crinane

alkaloids from Boophone disticha (Amaryllidaceae). J. Ethnopharmacol. 140,405–408.

Citoglu, G., Tanker, M., Gamusel, B., 1998. Antiinflammatory effects of lycorine andhaemanthidine. Phytother. Res. 12, 205–206.

Cooke, J.K., Warren, F.L., 1953. The alkaloids of the Amaryllidaceae. The chemicalindividuality of haemanthine. J. S. Afr. Chem. Inst. 6, 2–3.

Cragg, G.M., Newman, D.J., 2005. Plants as a source of anti-cancer agents. J.Ethnopharmacol. 100, 72–79.

Crouch, S.P.M., Kozlowski, R., Slater, K.J., Fletcher, J., 1993. The use of ATPbioluminescence as a measure of cell proliferation and cytotoxicity. J. Immunol.Methods 160, 81–88.

Dahlgren, R.M.T., Clifford, H.T., Yeo, P.F., 1985. The Families of the Monocotyledons.Springer, Berlin.

De Beer, J.J.J., Van Wyk, B.-E., 2011. An ethnobotanical survey of the Agter-Hantam,Northern Cape Province, South Africa. S. Afr. J. Bot. 77, 741–754.

De Leo, P., Dalessandro, G., De Santis, A., Arrigoni, O., 1973. Inhibitory effect oflycorine on cell division and cell elongation. Plant Cell Physiol. 14, 481–486.

De Smet, P.A.G.M., 1996. Some ethnopharmacological notes on African hallucino-gens. J. Ethnopharmacol. 50, 141–146.

De Smet, P.A.G.M., 1998. Traditional pharmacology and medicine in Africa.Ethnopharmacological themes in sub-Saharan art objects and utensils. J.Ethnopharmacol. 63, 1–179.

Dold, A.P., Cocks, M.L., 2002. The trade in medicinal plants in the Eastern CapeProvince, South Africa. S. Afr. J. Sci. 98, 589–597.

J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–2624

Du Plooy, W.J., Swart, L., Van Huysteen, G.W., 2001. Poisoning with Boophanedisticha: a forensic case. Hum. Exp. Toxicol. 20, 277–278.

Dyer, R.A., 1950. A review of the genus Brunsvigia. Plant Life 6, 63–83.Elgorashi, E.E., Malan, S.F., Stafford, G.I., Van Staden, J., 2006a. Quantitative

structure-activity relationship studies on acetylcholinesterase enzyme inhibi-tory effects of Amaryllidaceae alkaloids. S. Afr. J. Bot. 72, 224–231.

Elgorashi, E.E., Stafford, G.I., Jäger, A.K., Van Staden, J., 2006b. Inhibition of [3H]citalopram binding to the rat brain serotonin transporter by Amaryllidaceaealkaloids. Planta Med. 72, 1–3.

Elgorashi, E.E., Stafford, G.I., Van Staden, J., 2004. Acetylcholinesterase enzymeinhibitory effects of Amaryllidaceae alkaloids. Planta Med. 70, 260–262.

Elgorashi, E.E., Zschocke, S., Van Staden, J., 2003. The anti-inflammatory andantibacterial activities of Amaryllidaceae alkaloids. S. Afr. J. Bot. 69, 448–449.

Elisha, I.L., Elgorashi, E.E., Hussein, A.A., Duncan, G., Eloff, J.N., 2013. Acetylcholi-nesterase inhibitory effects of the bulb of Ammocharis coranica (Amaryllida-ceae) and its active constituent lycorine. S. Afr. J. Bot. 85, 44–47.

Evidente, A., Andolfi, A., Abou-Donia, A.H., Touema, S.M., Hommoda, H.M., Shawky,E., Motta, A., 2004. (�)-Amarbellisine, a lycorine-type alkaloid from Amaryllisbelladonna L. growing in Egypt. Phytochemistry 65, 2113–2118.

Evidente, A., Kornienko, A., 2009. Anticancer evaluation of structurally diverseAmaryllidaceae alkaloids and their synthetic derivatives. Phytochem. Rev. 8,449–459.

Fales, H.M., Wildman, W.C., 1961. Structure and sterochemistry of buphanamine. J.Org. Chem. 26, 881–886.

Fennell, C.W., Van Staden, J., 2001. Crinum species in traditional and modernmedicine. J. Ethnopharmacol. 78, 15–26.

Foden, A.P., 2010. Which witch doctor? The South African experience. DarlingtonCty Durh. Med. J. 3, 45–51.

Fouche, G., Cragg, G.M., Pillay, P., Kolesnikova, N., Maharaj, V.J., Senabe, J., 2008. Invitro anticancer screening of South African plants. J. Ethnopharmacol. 119,455–461.

Fouche, G., Khorombi, E., Kolesnikova, N., Maharaj, V.J., Nthambeleni, R., Van derMerwe, M., 2006. Investigation of South African plants for anticancer proper-ties. Pharmacologyonline 3, 494–500.

Gadaga, L.L., Tagwireyi, D., Dzangare, J., Nhachi, C.F.B., 2010. Acute oral toxicity andneurobehavioural toxicological effects of hydroethanolic extract of Boophonedisticha in rats. Hum. Exp. Toxicol. 30, 972–980.

Ghosal, S., 1986. Chemical constituents of Amaryllidaceae. Part 24. Crinafoline andcrinafolidine, two antitumor alkaloids from Crinum latifolium. J. Chem. Res. (S),312–313.

Gomes, N.G.M., Campos, M.G., Órfão, J.M.C., Ribeiro, C.A.F., 2009. Plants withneurobiological activity as potential targets for drug discovery. Prog. Neurop-sychopharmacol. Biol. Psychiatry 33, 1372–1389.

Goosen, A., Warren, F.L., 1957. Identity of haemanthine with buphanitine. Chem.Ind. (Lond.), 267.

Graham, J.G., Quinn, M.L., Fabricant, D.S., Farnsworth, N.R., 2000. Plants usedagainst cancer—an extension of the work of Jonathan Hartwell. J. Ethnophar-macol. 73, 347–377.

Greenblatt, H.M., Kryger, G., Lewis, T., Silman, I., Sussman, J.L., 1999. Structure ofacetylcholinesterase complexed with (�)-galanthamine at 2.3 Å resolution.FEBS Lett. 463, 321–326.

Greig, N.H., Mattson, M.P., Perry, T., Chan, S.L., Giordano, T., Subamurti, K., Rogers, J.T., Ovadia, H., Lahir, D.K., 2004. New therapeutic strategies and drug candidatesfor neurodegenerative diseases. Proc. N. Y. Acad. Sci. 1035, 290–315.

Grierson, D.S., Afolayan, A.J., 1999. An ethnobotanical study of plants used for thetreatment of wounds in the Eastern Cape. South Africa. J. Ethnopharmacol. 67,327–332.

Griffin, C., Sharda, N., Sood, D., Nair, J.J., McNulty, J., Pandey, S., 2007. Selectivecytotoxicity of pancratistatin-related natural Amaryllidaceae alkaloids: evalua-tion of the activity of two new compounds. Cancer Cell Int. 7, 10–16.

Guengerich, F.P., 1997. Role of cytochrome P450 enzymes in drug–drug interac-tions. Adv. Pharmacol. 43, 7–35.

Harvey, A.L., 1995. The pharmacology of galanthamine and its analogues. Pharma-col. Ther. 68, 113–128.

Hauth, H., Stauffacher, D., 1961. Die alkaloide von Buphane disticha (L.f.) Herb. Helv.Chim. Acta 44, 491–502.

Heinrich, M., Teoh, H.L., 2004. Galanthamine from snowdrop—the development of amodern drug against Alzheimer's disease from local Caucasian knowledge. J.Ethnopharmacol. 92, 147–162.

Heyman, H.M., Hussein, A.A., Meyer, J.J.M., Lall, N., 2009. Antibacterial activity ofSouth African medicinal plants against methicillin resistant Staphylococcusaureus. Pharmaceut. Biol. 47, 67–71.

Houghton, P.J., Ren, Y., Howes, M.J., 2006. Acetylcholinesterase inhibitors fromplants and fungi. Nat. Prod. Rep. 23, 181–199.

Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A.B., 1996. Zulu Medicinal Plants:An Inventory. University of Natal Press, Pietermaritzburg.

Hutchings, A., Van Staden, J., 1994. Plants used for stress-related ailments intraditional Zulu, Xhosa and Sotho Medicine. Part 1: plants used for headaches. J.Ethnopharmacol. 43, 89–124.

Ieven, M., Van den Berghe, D.A., Vlietinck, A.J., 1983. Plant antiviral agents. IV.Influence of lycorine on growth pattern of three animal viruses. Planta Med. 49,109–114.

Ieven, M., Vlietinck, A.J., Van den Berghe, D.A., Totte, J., Dommisse, R., Esmans, E.,Alderweireldt, F., 1982. Plant antiviral agents. III. Isolation of alkaloids fromClivia miniata Regel (Amaryllidaceae). J. Nat. Prod. 45, 564–573.

Jäger, A.K., Hutchings, A., Van Staden, J., 1996. Screening of Zulu medicinal plantsfor prostaglandin-synthesis inhibitors. J. Ethnopharmacol. 52, 95–100.

Jimenez, A., Santos, A., Alonso, G., Vazquez, D., 1976. Inhibitors of protein synthesisin eukaryotic cells. Comparative effects of some Amaryllidaceae alkaloids.Biochim. Biophys. Acta 425, 342–348.

Jin, Z., 2011. Amaryllidaceae and Sceletium alkaloids. Nat. Prod. Rep. 28, 1126–1142.Kornienko, A., Evidente, A., 2008. Chemistry, biology and medicinal potential of

narciclasine and its congeners. Chem. Rev. 108, 1982–2014.Kretzing, S., Abraham, G., Seiwert, B., Ungemach, F.R., Krügel, U., Regenthal, R., 2011.

Dose-dependent emetic effects of the Amaryllidaceous alkaloid lycorine inbeagle dogs. Toxicon 57, 117–124.

Lagarde, E., Dirk, T., Puren, A., Reathe, R.T., Bertran, A., 2003. Acceptability of malecircumcision as a tool for preventing HIV infection in a highly infectedcommunity in South Africa. AIDS 17, 89–95.

Laing, R.O., 1979. Three cases of poisoning by Boophane disticha. Cent. Afr. J. Med.25, 265–266.

Lamoral-Theys, D., Decaestecker, C., Mathieu, V., Dubois, J., Kornienko, A., Kiss, R.,Evidente, A., Pottier, L., 2010. Lycorine and its derivatives for anticancer drugdesign. Mini-Rev. Med. Chem. 10, 41–50.

Lewin, L., 1912a. Studies on Buphane disticha (Haemanthus toxicarius). Arch. Exp.Pathol. Pharmakol. 68, 333–340.

Lewin, L., 1912b. Über haemanthin. Arch. Exp. Pathol. Pharmakol. 70, 302.Li, S., Chen, C., Zhang, H., Guo, H., Wang, H., Wang, L., Zhang, X., Hua, S., Yu, J., Xiao,

P., Li, Y., Liu, J., Tang, L., Shi, Y., Ren, W., Hu, W., 2007. Apoptosis induced bylycorine in KM3 cells is associated with the G0/G1 cell cycle arrest. Oncol. Rep.17, 377–384.

Likhitwitayawuid, K., Angerhofer, C.K., Chai, H., Pezzuto, J.M., Cordell, G.A., Ruan-grungsi, N., 1993. Cytotoxic and antimalarial alkaloids from the bulbs of Crinumamabile. J. Nat. Prod. 56, 1331–1338.

Liu, J., Hu, W., He, L., Ye, M., Li, Y., 2004. Effects of lycorine on HL-60 cells viaarresting cell cycle and inducing apoptosis. FEBS Lett. 578, 245–250.

Liu, J., Li, Y., Tang, L.J., Zhang, G.P., Hu, W.X., 2007. Treatment of lycorine on SCIDmice model with human APL cells. Biomed. Pharmacother. 61, 229–234.

Lopez, S., Bastida, J., Viladomat, F., Codina, C., 2002. Acetylcholinesterase inhibitoryactivity of some Amaryllidaceae alkaloids and Narcissus extracts. Life Sci. 71,2521–2529.

Louw, C.A.M., Regnier, T.J.C., Korsten, L., 2002. Medicinal bulbous plants of SouthAfrica and their traditional relevance in the control of infectious diseases. J.Ethnopharmacol. 82, 147–154.

Mabona, U., Van Vuuren, S.F., 2013. Southern African medicinal plants used to treatskin diseases. S. Afr. J. Bot. 87, 175–193.

McNulty, J., Nair, J.J., Bastida, J., Pandey, S., Griffin, C., 2009a. Structure-activitystudies on the lycorine pharmacophore: a potent inducer of apoptosis inhuman leukemia cells. Phytochemistry 70, 913–919.

McNulty, J., Nair, J.J., Singh, M., Crankshaw, D.J., Holloway, A.C., Bastida, J., 2009b.Selective cytochrome P450 3A4 inhibitory activity of Amaryllidaceae alkaloids.Bioorg. Med. Chem. Lett. 19, 3233–3237.

McNulty, J., Nair, J.J., Codina, C., Bastida, J., Pandey, S., Gerasimoff, J., Griffin, C., 2007.Selective apoptosis-inducing activity of crinum-type Amaryllidaceae alkaloids.Phytochemistry 68, 1068–1074.

McNulty, J., Nair, J.J., Little, J.R.L., Brennan, J.D., Bastida, J., 2010. Structure–activityrelationship studies on acetylcholinesterase inhibition in the lycorine series ofAmaryllidaceae alkaloids. Bioorg. Med. Chem. Lett. 20, 5290–5294.

Mebs, D., Röhrich, J., Kauert, G., Becker, P.-R., 1996. Pfeilgifte. Eine toxicologischespurensuche im museum. Dtsch. Apoth. Ztg. 136, 2062–2065.

Meerow, A.W., Clayton, J.R., 2004. Generic relationships among the baccate-fruitedAmaryllidaceae (tribe Haemantheae) inferred from plastid and nuclear non-coding DNA sequences. Plant System. Evol. 244, 141–155.

Meerow, A.W., Fay, M.F., Guy, C.L., Li, Q.B., Zaman, F.G., Chase, M.W., 1999.Systematics of Amaryllidaceae based on cladistic analysis of plastid RBCL andTRNL-F sequence data. Am. J. Bot. 86, 1325–1345.

Meerow, A.W., Snijman, D.A., 1998. Amaryllidaceae. In: Kubitzki, K. (Ed.), TheFamilies and Genera of Vascular Plants, vol. 3. Springer, Berlin, pp. 83–110.

Meerow, A.W., Snijman, D.A., 2001. Phylogeny of Amaryllidaceae tribe Amaryllideaebased on NRDNA ITS sequences and morphology. Am. J. Bot. 88, 2321–2330.

Monton, M.R.N., Lebert, J.M., Little, J.R.L., Nair, J.J., McNulty, J., Brennan, J.D., 2010. Asol–gel derived acetylcholinesterase microarray for nanovolume small-molecule screening. Anal. Chem. 82, 9365–9373.

Müller-Doblies, D., Müller-Doblies, U., 1996. Tribes and subtribes and some speciescombinations in Amaryllidaceae J. St.-Hil. Emend R. Dahlgren & al. 1985. Fed.Rep. 107, 1–9. (S.c).

Mutseura, M., Tagwireyi, D., Gadaga, L.L., 2013. Pre-treatment of BALB/C mice with acentrally acting serotonin antagonist (Cyproheptadine) reduces mortality fromBoophone disticha poisoning. Clin. Toxicol. 51, 16–22.

Nair, J.J., Aremu, A.O., Van Staden, J., 2011. Isolation of narciprimine from Cyrtanthuscontractus (Amaryllidaceae) and evaluation of its acetylcholinesterase inhibi-tory activity. J. Ethnopharmacol. 137, 1102–1106.

Nair, J.J., Bastida, J., Codina, C., Viladomat, F., Van Staden, J., 2013. Alkaloids of theSouth African Amaryllidaceae: a review. Nat. Product Commun. 8, 1335–1350.

Nair, J.J., Bastida, J., Viladomat, F., Van Staden, J., 2012a. Cytotoxic agents of thecrinane series of Amaryllidaceae alkaloids. Nat. Product Commun. 7,1677–1688.

Nair, J.J., Campbell, W.E., Gammon, D.W., Albrecht, C.F., Viladomat, F., Codina, C.,Bastida, J., 1998. Alkaloids from Crinum delagoense. Phytochemistry 49,2539–2543.

J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 25

Nair, J.J., Manning, J.C., Van Staden, J., 2012b. Distichamine, a chemotaxonomicmarker for the genus Boophone Herb. (Amaryllidaceae). S. Afr. J. Bot. 83, 89–91.

Nair, J.J., Rárová, L., Strnad, M., Bastida, J., Van Staden, J., 2012c. Apoptosis-inducingeffects of distichamine and narciprimine, rare alkaloids of the plant familyAmaryllidaceae. Bioorg. Med. Chem. Lett. 22, 6195–6199.

Nair, J.J., Van Staden, J., 2012. Acetylcholinesterase inhibition within the lycorineseries of Amaryllidaceae alkaloids. Nat. Product Commun. 7, 959–962.

Neergaard, J.S., Andersen, J., Pedersen, M.E., Stafford, G.I., Van Staden, J., Jäger, A.K.,2009. Alkaloids from Boophone disticha with affinity to the serotonin transpor-ter. S. Afr. J. Bot. 75, 371–374.

Neuwinger, H.D., 1994. African Ethnobotany: Poisons and Drugs. Chapman andHall, Germany.

Neuwinger, H.D., Mebs, D., 1997. Boophone disticha. A hallucinogenic African plant.Dtsch. Apoth. Ztg. 137, 1127–1132.

Nielsen, N.D., Sandager, M., Stafford, G.I., Van Staden, J., Jäger, A.K., 2004. Screeningof indigenous plants from South Africa for affinity to the serotonin reuptaketransport protein. J. Ethnopharmacol. 94, 159–163.

Nyazema, N.Z., 1986. Herbal toxicity in Zimbabwe. Trans. R. Soc. Trop. Med. Hyg. 80,448–450.

Olivier, W., 1980. The genus Cyrtanthus Ait. Veld Flora (Kirstenbosch) 66, 78–81.Pan, L., Chai, H., Kinghorn, A.D., 2010. The continuing search for antitumor agents

from higher plants. Phytochem. Lett. 3, 1–8.Pedersen, M.E., Szewczyk, B., Stachowicz, K., Wieronska, J., Andersen, J., Stafford, G.

I., Van Staden, J., Pilc, A., Jäger, A.K., 2008. Effects of South African traditionalmedicine in animal models for depression. J. Ethnopharmacol. 119, 542–548.

Pettit, G.R., Gaddamidi, V., Goswami, A., Cragg, G.M., 1984. Antineoplastic agents.99. Amaryllis belladonna. J. Nat. Prod. 47, 796–801.

Pettit, G.R., Pettit III, G.R., Backhaus, R.A., Boyd, M.R., Meerow, A.W., 1993. Cellgrowth inhibitory isocarbostyrils from Hymenocallis. J. Nat. Prod. 56,1682–1687.

Philander, L.A., 2011. An ethnobotany of Western Cape rasta bush medicine. J.Ethnopharmacol. 138, 578–594.

Philippe, G., Angenot, L., 2005. Recent developments in the field of arrow and dartpoisons. J. Ethnopharmacol. 100, 85–91.

Pote, W., Tagwireyi, D., Chinyanga, H.M., Musara, C., Nyandoro, G., Chifamba, J.,Nkomozepi, P., 2013. Cardiovascular effects of Boophone disticha aqueousethanolic extract on early maternally separated BALB/C mice. J. Ethnopharma-col. 148, 379–385.

Pryce, C.R., Feldon, J., 2003. Long-term neurobehavioural impact of the postnatalenvironment in rats: manipulations, effects and mediating mechanisms.Neurosci. Biobehav. Rev. 27, 57–71.

Rabe, T., Van Staden, J., 1997. Antibacterial activity of South African plants used formedicinal purposes. J. Ethnopharmacol. 56, 81–87.

Risa, J., Risa, A., Adsersen, A., Gauguin, B., Stafford, G.I., Van Staden, J., Jäger, A.K.,2004a. Screening of plants used in southern Africa for epilepsy and convulsionsin the GABAA–benzodiazepine receptor assay. J. Ethnopharmacol. 93, 177–182.

Risa, A., Risa, J., Adsersen, A., Stafford, G.I., Van Staden, J., Jäger, A.K., 2004b.Acetylcholinesterase inhibitory activity of plants used as memory-enhancers intraditional South African medicine. S. Afr. J. Bot. 70, 664–666.

Sandager, M., Nielsen, N.D., Stafford, G.I., Van Staden, J., Jäger, A.K., 2005. Alkaloidsfrom Boophone disticha with affinity to the serotonin transporter in rat brain. J.Ethnopharmacol. 98, 367–370.

Şener, B., Orhan, I., Stayavivad, J., 2003. Antimalarial activity screening of somealkaloids and the plant extracts from Amaryllidaceae. Phytother. Res. 17,1220–1223.

Shale, T.L., Stirk, W.A., Van Staden, J., 1999. Screening of medicinal plants used inLesotho for antibacterial and anti-inflammatory activity. J. Ethnopharmacol. 67,347–354.

Snijman, D.A., Linder, H.P., 1996. Phylogenetic relationships, seed characters, anddispersal system evolution in Amaryllideae (Amaryllidaceae). Ann. MissouriBot. Gard. 83, 362–386.

Sobiecki, J.F., 2002. A preliminary inventory of plants used for psychoactivepurposes in southern African healing traditions. Trans. R. Soc. S. Afr. 57, 1–24.

Sobiecki, J.F., 2008. A review of plants used in divination in southern Africa andtheir psychoactive effects. S. Afr. Hum. 20, 333–351.

Stafford, G.I., Pedersen, P.D., Jäger, A.K., Van Staden, J., 2007. Monoamine oxidaseinhibition by southern African traditional medicinal plants. S. Afr. J. Bot. 73,384–390.

Stafford, G.I., Pedersen, M.E., Van Staden, J., Jäger, A.K., 2008. Review on plants withCNS-effects used in traditional South African medicine against mental diseases.J. Ethnopharmacol. 119, 513–537.

Stahl, S.M., 1998. Mechanism of action of serotonin selective reuptake inhibitors:serotonin receptors and pathways mediate therapeutic effects and side effects.J. Affect. Disord. 51, 215–235.

Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., Silman, I., 1991.Atomic structure of acetylcholinesterase from Torpedo californica: a prototypicacetylcholine-binding protein. Science 253, 872–879.

Schwartz, T.L., Nihalani, N., 2006. Tiagabine in anxiety disorders. Expert Opin.Pharmacother. 7, 1977–1987.

Taylor, J.L.S., Elgorashi, E.E., Maes, A., Van Gorp, U., De Kimpe, N., Van Staden, J.,Verschaeve, L., 2003. Investigating the safety of plants used in South Africantraditional medicine: testing for genotoxicity in the micronucleus and alkalinecomet assays. Environ. Mol. Mutagen. 42, 144–154.

Traub, H.P., 1963. Amaryllid notes. Plant Life 19, 57–62.Tutin, F., 1911a. The constituents of the bulb of Buphane disticha. J. Chem. Soc. Trans.

99, 1240–1248.Tutin, F., 1911b. Constituents of the bulb of Buphane disticha. Proc. Chem. Soc. Lond.

27, 149.Tutin, F., 1913. Constituents of Buphane disticha. Arch. Exp. Pathol. Pharmakol. 69,

314.Van Goietsenoven, G., Andolfi, A., Lallemand, B., Cimmino, A., Lamoral-Theys, D.,

Gras, T., Abou-Donia, A., Dubois, J., Lefranc, F., Mathieu, V., Kornienko, A., Kiss,R., Evidente, A., 2010. Amaryllidaceae alkaloids belonging to different structuralsubgroups display activity against apoptosis-resistant cancer cells. J. Nat. Prod.73, 1223–1227.

Van Wyk, B.-E., 2008. A review of Khoi-San and Cape Dutch medical ethnobotany. J.Ethnopharmacol. 119, 331–341.

Van Wyk, B.-E., De Wet, H., Van Heerden, F.R., 2008. An ethnobotanical survey ofmedicinal plants in the southeastern Karoo, South Africa. S. Afr. J. Bot. 74,696–704.

Van Wyk, B.-E., Van Heerden, F., Van Oudtshoorn, B., 2005. Poisonous Plants ofSouth Africa. Briza Publications, Pretoria.

Van Wyk, B.-E., Van Oudtshoorn, B., Gericke, N., 2002. Medicinal Plants of SouthAfrica. Briza Publications, Pretoria.

Vargas, F.C., 1981. Plant motifs on Inca ceremonial drinking vessels from Peru. Bot. J.Linn. Soc. 82, 313–325.

Verschaeve, L., Van Staden, J., 2008. Mutagenic and antimutagenic properties ofextracts from South African traditional medicinal plants. J. Ethnopharmacol.119, 575–587.

Verzár, R., Petri, G., 1987. Medicinal plants in Mozambique and their popular use. J.Ethnopharmacol. 19, 67–80.

Viladomat, F., Bastida, J., Codina, C., Nair, J.J., Campbell, W.E., 1997. Alkaloids of theSouth African Amaryllidaceae. In: Pandalai, S.G. (Ed.), Recent Research Devel-opments in Phytochemistry, vol. 1. Research Signpost Publishers, Trivandrum,pp. 131–171.

Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants ofSouthern and Eastern Africa. Livingston Ltd, Edinburgh.

WHO, 2008. World Cancer Report 2008. WHO Press, Geneva, Switzerland.Wilcken, A., Keil, T., Dick, B., 2010. Traditional male circumcision in eastern and

southern Africa: a systematic review of prevalence and complications. Bull.World Health Organ. 88, 907–914.

Williams, V.L., Victor, J.E., Crouch, N.R., 2013. Red listed medicinal plants of SouthAfrica: status, trends, and assessment challenges. S. Afr. J. Bot., 86; , pp. 23–35.

Williamson, G., 2012. The remarkable Boophone disticha (L.f) Herb. (Amaryllida-ceae); a survivor from the northern tropical savanna woodlands to the SouthAfrican semi-arid Karoo. Cactus Succul. J. 84, 88–91.

Wintola, O.A., Afolayan, A.J., 2010. Ethnobotanical survey of plants used for thetreatment of constipation within Nkonkobe municipality of South Africa. Afr. J.Biotechnol. 9, 7767–7770.

Wrinkle, G., 1984. An introduction to the genus Boophane. Herbertia 40, 77–82.Yamada, M., Yasuhara, H., 2004. Clinical pharmacology of MAO inhibitors: safety

and future. Neurotoxicology 25, 215–221.

J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–2626

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