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Traditional usage, phytochemistry and pharmacology of the South African medicinal plant Boophone disticha (L.f...

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Review Traditional usage, phytochemistry and pharmacology of the South African 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 article info abstract

Article history: Ethnopharmacological relevance: Boophone disticha is the most common member of the South African Received 2 September 2013 Amaryllidaceae used extensively in traditional medicine of the various indigenous population groups, Received in revised form including the Sotho, Xhosa and Zulu as well as the San. This survey was carried out to identify and 23 October 2013 highlight areas relevant to the traditional usage of Boophone disticha. Pharmacological aspects were Accepted 23 October 2013 examined with the purpose of reconciling these with the traditional usage of the plant. In relation to Available online 7 November 2013 phytochemical make-up, particular attention was paid on how its alkaloid constitution might corroborate Keywords: the various biological effects manifested by the plant. Alkaloid Materials and methods: Information gathering involved the use of four different database platforms, Amaryllidaceae including Google Scholar, ScienceDirect, SciFinders and Scopus. Arrangement and detailing of this Boophone disticha information is as reflected in the various sections of the paper. Traditional medicine Results: Sixteen categories were identified under which Boophone disticha finds use in traditional medicine. 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 the circulatory, gastrointestinal, muscular, neurological, respiratory and urinary systems. The four remaining categories relate to use for inflammatory conditions, cancer, malaria and tuberculosis. Overall, three areas were discernible in which Boophone disticha finds most usage, which are (i) ailments pertaining to the CNS, (ii) wounds and infections, and (iii) inflammatory conditions. In addition, several aspects pertaining to 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 for the Amaryllidaceae and its relevance to South African traditional medicine. Furthermore, its promising pharmacological and phytochemical profiles have stimulated significant interest in the clinical realm, especially in the areas of cancer and motor neuron disease chemotherapy. These collective properties should prove useful in steering the progress of the plant towards a wider audience. & 2013 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction...... 13 2. Description and distribution ...... 14 3. Taxonomy...... 15 4. Conservation status ...... 15 5. Traditional usage ...... 15 6. Phytochemistry ...... 18 6.1. Alkaloids of the South African Amaryllidaceae ...... 18

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: [email protected] (J. Van Staden).

6.2. Alkaloids from Boophone disticha ...... 19 7. Pharmacology ...... 19 7.1. Antimicrobial activity ...... 19 7.2. Anti-inﬂammatory activity ...... 20 7.3. Effects on the central nervous system ...... 20 7.3.1. Hallucinogenic effects ...... 20 7.3.2. Mental disorders ...... 20 7.4. Anticancer activity...... 22 7.5. Toxicological studies ...... 23 8. Conclusions ...... 24 Acknowledgements ...... 24 References...... 24

1. Introduction et al., 2013). Chief amongst these secondary metabolites is the alkaloid galanthamine 1 (also referred to as ‘galantamine’) The Amaryllidaceae J. St.-Hil. is a large family of bulbous (Scheme 1), a prescription drug in the treatment of Alzheimer's flowering plants comprising roughly 1000 species in 79 genera disease (AD) (Greig et al., 2004; Heinrich and Teoh, 2004; with a pantropical distribution (Meerow and Snijman, 1998). Houghton et al., 2006). Having captured a significant share of Andean South America, the Mediterranean and South Africa have the multi-billion dollar global market over the past decade, been identified as centres of prominence for these monocotyle- galanthamine, like other acetylcholinesterase (AChE) inhibitor donous plants (Meerow and Snijman, 1998). Their sphere of regimens, remains steadfast as a primary choice in the chemother- influence extends into areas as diverse as agriculture, horticulture, apeutic approaches to AD (Greig et al., 2004; Heinrich and Teoh, ethnobotany, traditional medicine (TM) and pharmacology 2004; Houghton et al., 2006). This status is due chiefly to its (Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013). For potent, selective and reversible inhibitory interaction with AChE example, daffodils (Narcissus) are amongst the most commonly which is known to play a significant role in the progression of sold cut-flower varieties and are thus of significant importance to neurodegeneration associated with the pathophysiology of AD the floriculture industry (Bastida et al., 2006). Furthermore, (Greig et al., 2004; Heinrich and Teoh, 2004; Houghton et al., several members of the family have intensive usage in the 2006). The research and development of galanthamine into a traditional medicinal practices of indigenous peoples around the viable contemporary AD drug owes much to TM knowledge world (Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013). evolving out of the usage of Galanthus species, from which The long history of association of the Amaryllidaceae with TM in galanthamine was first isolated, in the Caucasus region of Eastern the Mediterranean basin can be traced back to the times of Europe (Heinrich and Teoh, 2004). Hippocrates and Pliny who recorded diverse medicinal applica- In terms of structural diversity, Amaryllidaceae alkaloids have tions of Narcissus (Pettit et al., 1993; Kornienko and Evidente, been classified into six distinct groups (Viladomat et al., 1997; 2008). Furthermore, archaeological findings at purportedly Inca Bastida et al., 2006; Jin, 2011; Nair et al., 2013). Galanthamine 1, ruins in South America have indicated that Ismene, Pyolirion and lycorine 2 and crinine 3 are representative of the three major Stenomesson as depicted on ceremonial drinking vessels may have structural-types for these alkaloids (Scheme 1), while homolycor- been a part of popular culture (Vargas, 1981), while San rock ine 4, tazettine 5 and montanine 6 make up the minor series of paintings of Boophone and Brunsvigia species in South Africa attest compounds discernible within the Amaryllidaceae (Viladomat to the usage of the Amaryllidaceae by the region's first inhabitants et al., 1997; Bastida et al., 2006; Jin, 2011; Nair et al., 2013). Other (Dyer, 1950; Van Wyk, 2008). less-conspicuous members include degraded, oxidised and trun- Roughly one-third of the Amaryllidaceae is found in South cated variants such as trisphaeridine 7 and ismine 8 (Viladomat Africa, the majority of which are distributed through the Capensis et al., 1997; Bastida et al., 2006; Jin, 2011; Nair et al., 2013). All floral kingdom of the Western Cape (Meerow and Snijman, 1998). these structures are related as a consequence of their biogenesis Biosystematically, members of the South African Amaryllidaceae from the common amino acid derived precursor norbelladine 9 are adherent almost exclusively to the tribes Amaryllideae, Hae- (Viladomat et al., 1997; Bastida et al., 2006; Jin, 2011; Nair et al., mantheae and Cyrtantheae which are three of the 14 recognised 2013). tribes in this family (Olivier, 1980; Snijman and Linder, 1996; In addition to the therapeutic success of galanthamine in the Meerow and Clayton, 2004). Amaryllideae J. St.-Hil. is a mono- motor neuron disease (MND) arena, other clinical candidates have phyletic group consisting of 11 currently recognised genera and been identified in the Amaryllidaceae with significant potential for approximately 155 species (Snijman and Linder, 1996). Members of drug development. Of these, the phenanthridones (such as pan- the tribe, typified by Crinum, inhabit grassland, savannah and cratistatin 10) of the lycorine 2 series have attracted most interest tropical forests in sub-Saharan Africa, but are most speciose in as anticancer agents due to their potent and cell line-specific South Africa where habitats include semi-arid dwarf shrublands in antiproliferative activities and it has been suggested that a the winter rainfall region (Snijman and Linder, 1996). By contrast, commercial target should be made available within the next the tribe Haemantheae Pax (Hutchinson) consists of five genera decade (Kornienko and Evidente, 2008). Thus, with galanthamine (Scadoxus, Haemanthus, Clivia, Gethyllis and Apodolirion) which are 1 and pancratistatin 10 entrenched as veritable leads, the Amar- distributed through the various regions in South Africa (Meerow yllidaceae provides a vast resource platform for drug discovery in and Clayton, 2004). Of the African clades, Cyrtantheae Salisb. is the cancer, MND and allied disciplines (Viladomat et al., 1997; recognised as a tribe unto itself consisting of a single endemic Bastida et al., 2006; Nair et al., 2013). genus, Cyrtanthus (Olivier, 1980). Given these interesting biological traits, it is not surprising that Apart from its morphological and floral characteristics, the the Amaryllidaceae is one of the most common bulbous plants Amaryllidaceae is widely known for its unique alkaloid constitu- exploited on a global scale in both traditional and mainstream ents (Viladomat et al., 1997; Bastida et al., 2006; Jin, 2011; Nair sectors of healthcare (Viladomat et al., 1997; Bastida et al., 2006; 14 J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26

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

Fennell and Van Staden, 2001; Nair et al., 2013). This is also true Snijman, 1998; Williamson, 2012). Now exclusively referred to for the local scene where Amaryllidaceae plants find heavy usage as Boophone disticha, it is distributed along East Africa, ranging in TM and are consistently amongst the most popular bulbous from Sudan in the north to the Western Cape Province in the plants traded on such markets (Hutchings et al., 1996; Louw et al., south. In South Africa it is widely occurring with a range covering 2002; Brueton et al., 2008). Amongst the South African Amarylli- the northern tropical savannah woodlands to the winter rainfall daceae contingent, Boophone disticha stands out as the most region of the Western Cape, including the desert-like Karoo common plant used in both the formal and informal sectors of biome as well as the Eastern seaboard (Wrinkle, 1984). By TM, as well as in local research initiatives (Hutchings et al., 1996; contrast, Boophone haemanthoides is a rare and threatened Dold and Cocks, 2002; Louw et al., 2002; Brueton et al., 2008). species with a restricted territory within the winter rainfall However, the plant is also known to be one of the several toxic region of South Africa and parts of southern Namibia (Wrinkle, species of the Amaryllidaceae (Van Wyk et al., 2002, 2005). Thus, 1984). The generic name ‘Boophone’ is derived from the Greek within the context of South African TM details of the traditional ‘bous’ (ox) and ‘phone’ (death) while the specificname‘disticha’ uses, phytochemistry and pharmacology of Boophone disticha are refers to the leaves which are erect in a fan shape. Also known as afforded herein with the aim of reconciling the traditional aspects ‘sore-eye flower’ (‘seerooglelie’ in Afrikaans or ‘incotho’ in of its usage with more recent discoveries in the pharmacology of Xhosa), Boophone disticha is an attractive, deciduous plant which the Amaryllidaceae. flowers between July and October (Wrinkle, 1984). The large bulbs are covered with a thick layer of papyraceous scales, the skeletal remains of long dried leaves, which rise above soil level 2. Description and distribution and serve to protect the developing flower and newly emerged leaves (Williamson, 2012). Individual leaves twist slightly along The genus Boophone Herb. comprises of only two species: their length and are greyish-green to dark green in colour Boophone disticha (L.f.) Herb. and Boophone haemanthoides F.M. (Williamson, 2012)(Fig. 1). The peduncle is stout and carries a Leight. (Meerow and Snijman, 1998). Boophone disticha has at dense, rounded ball of numerous red-pink to dark pink flowers various instances previously been referred to as ‘Amaryllis dis- with long pedicels which elongate even further and stiffen during ticha’, ‘Boophane disticha’, ‘Boophone toxicaria’, ‘Buphane disticha’ maturity to carry the dried seed capsules (Williamson, 2012) and ‘Haemanthus toxicarius’ (Wrinkle, 1984; Meerow and (Fig. 1). J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 15

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

3. Taxonomy its medicinally significant alkaloid constituents, it is not surprising that these plants feature prominently amongst the manifold Phylogenetic relationships within the Amaryllidaceae have challenges posed to the conservation sector. This has been further come under intense scrutiny over the past several decades and exacerbated by more recent concerns such as unsustainable usage, significant efforts have been directed towards the African tribes unscrupulous collection methods as well as habitat destruction. Amaryllideae, Haemantheae and Cyrtantheae (Traub, 1963; Olivier, A recent review of red-listed medicinal plants of South Africa has 1980; Dahlgren et al., 1985; Müller-Doblies and Müller-Doblies, shown that 42 species of the Amaryllidaceae have been documen- 1996; Snijman and Linder, 1996; Meerow and Snijman, 1998; ted in TM, representing 2% of the total medicinal flora (Williams Meerow et al., 1999; Meerow and Snijman, 2001; Meerow and et al., 2013). However, of greater concern is that 26% of the total Clayton, 2004). As early as 1963, Traub proposed a two-tier local Amaryllid population has been classified as ‘threatened’ or structure for the tribe Amaryllideae based on both vegetative ‘near threatened’ (Williams et al., 2013). The status of Boophone and floral morphological characteristics. Accordingly, subtribe disticha has been indexed as ‘declining’ (Williams et al., 2013) Crineae (Ammocharis Herb., Boophone Herb., Brunsvigia Heist., which is not unexpected given the extent of its exploitation in TM Crinum L., Cybistetes Milne-Redh. & Schweick. and Nerine Herb.) (Dold and Cocks, 2002). To this end, advances in seed and was distinguished from subtribe Strumarieae (Carpolyza Salisb., molecular biology as well as the biotechnology arena have expe- Hessea Herb. and Strumaria Jacq.). Contrary to this subtribal dited the availability of propagated plants at TM markets to bolster classification, Dahlgren et al. (1985) combined all nine genera stocks under threat from unsustainable practices (Cheesman, listed above together with Amaryllis L. into the single tribe 2013). Amaryllideae. The 1996 classification of Müller-Doblies and Müller-Doblies re-introduces the subtribal structure to the tribe Amaryllideae but in this case with four component subtribes: 5. Traditional usage (i) Amaryllidinae (Amaryllis, Namaquanula D. and U. M-D. and Nerine); (ii) Boophoninae (Boophone, Brunsvigia and Crossyne The fact that Boophone disticha can be traced back via rock Salisb.); (iii) Strumariinae (Bokkeveldia D. and U. M-D., Carpolyza, paintings to the original inhabitants of South Africa suggests a long Dewinterella D. and U. M-D., Gemmaria D. and U. M-D., Hessea, history of association of the plant with the traditional practices of Strumaria and Tedingea D. and U. M-D.); and (iv) Crininae (Crinum, the indigenous peoples of the region (Van Wyk, 2008). This is Ammocharis and Cybistetes). The revision of the tribe Amaryllideae further substantiated by the discovery of the 2000 year-old by Snijman and Linder (1996) is widely accepted as the most remains of a Khoi-San inhabitant of the Kouga/Baviaanskloof area comprehensive classification to date and reverts back to the initial of the Eastern Cape, found mummified with Boophone disticha two-tier structure of Traub (1963) with minor adjustments. Based scales (Binneman, 1999; Van Wyk, 2008). Furthermore, records of on morphological, anatomical and cytological data, the tribe was usage of the plant by Cape-Dutch settlers (Watt and Breyer- resolved into two monophyletic subtribes: (i) Crininae comprising Brandwijk, 1962) suggest that the application of indigenous Ammocharis, Boophone, Crinum and Cybistetes; and (ii) Amaryllidi- knowledge (IK) may have been essential to the medicinal needs nae comprising Amaryllis, Brunsvigia, Carpolyza, Crossyne, Hessea, of early settlers to the colony. ‘Khoi-San’ is a unifying name for the Nerine and Strumaria (Snijman and Linder, 1996). Thus, based on ‘Khoi’ and ‘San’ tribes of southern Africa, two ethnic groups who these characteristics as well as evidence gleaned from DNA share physical and linguistic characteristics distinct from the Bantu sequence data (Meerow and Snijman, 2001), the placement of majority of the region (Barnard, 1992). Usage of the Amaryllida- the genus Boophone in the tribe Amaryllideae has never been ceae for medicinal purposes in South Africa is extensive which is in doubt. reflected by the popularity of these plants on traditional markets of the three most populous tribes of the region (the Sotho, Xhosa and Zulu) (Hutchings et al., 1996; Dold and Cocks, 2002; Louw 4. Conservation status et al., 2002). For example, Hutchings et al. (1996) documented the usage of 13 different Amaryllid species by traditional healers from Given the natural abundance of the Amaryllidaceae in South various districts within the Zululand area. Of these, eight were for Africa, its widespread usage in TM, its ornamental value as well as urinary or venereal diseases; six for headache and fever; six for 16 J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 swelling, growths and joint ailments; five for skin conditions, knowledge of the bulbs in selecting as more toxic those growing bruises, sprains and fractures; four for respiratory problems; three under cover of shade over those in sunnier spots (Watt and Breyer- for gastrointestinal disorders; and two for use as internal purifiers Brandwijk, 1962). Evidence for this essential cultural practice (Hutchings et al., 1996). Furthermore, a survey by Dold and Cocks amongst the San can be seen in arrow heads, known to have been (2002) covering six urban centres in the Eastern Cape Province treated with Boophone disticha and taken from the Cape to Berlin showed that a minimum of 166 medicinal plants were traded on in 1806, from which a substance (“haemanthine”) was isolated these markets of the indigenous Xhosa, utilising 525 tonnes of over one hundred years later (Lewin, 1912a,b; Watt and Breyer- plant material with an estimated value of $3 million annually. In Brandwijk, 1962). Although no trace of the compound remained the study, Boophone disticha was listed amongst the most fre- for comparative studies several decades later, it was nonetheless quently traded plants at these centres (Dold and Cocks, 2002). reputed to be either buphanamine 11 or nerbowdine 12 (Bates Another study revealed that Boophone disticha is one of the most et al., 1957; Goosen and Warren, 1957; Fales and Wildman, 1961), commonly traded bulbous plants at the Faraday ‘umuthi’ market in two known alkaloid constituents of Boophone disticha (refer to Johannesburg (the largest TM market in South Africa) (Brueton Section 6.2). Furthermore, an arrow belonging to the San of the et al., 2008). Given these facts, it is clear that Boophone disticha is Kalahari, known to have been in possession of the Völkerkunde the most common of the Amaryllidaceae members exploited Museum der Universität Göttingen since 1937, was shown to across many sectors of TM in South Africa. contain alkaloids characteristic of Boophone disticha 60 years later For the purpose of this survey, 16 categories (Table 1)were (Mebs et al., 1996; De Smet, 1998). An in-depth look at the identified under which Boophone disticha finds use in the TM phytochemical makeup of Boophone disticha follows in Section practices of indigenous people of the region. Furthermore, while 6.2 of this paper. bulbs were the most common plant part used in TM, leaves, Another area that Boophone disticha finds extensive usage is in flowers and roots were also utilised but to a lesser extent. In the practice of circumcision and initiation rituals (Table 1)ofthe broad terms, Boophone disticha is used for cultural (such as various indigenous tribes of South Africa (Watt and Breyer- adornment), health and well-being purposes (bulb tonic taken Brandwijk, 1962; Verzár and Petri, 1987; De Smet, 1996; for weakness) as well as for personal injury (wounds, cuts and Hutchings et al., 1996; Viladomat et al., 1997; Grierson and bruises) (Table 1, entries 1–3). Six categories relating to body Afolayan, 1999; Du Plooy et al., 2001; Van Wyk et al., 2008; system functions were identified, including usage for circulatory, Philander, 2011). Male circumcision is performed on adolescent gastrointestinal, muscular, neurological, respiratory, and urinary or young men for cultural reasons, particularly as an initiation ailments (Table 1, entries 4–9). Of significance in these categories ritual and a rite of passage into manhood (Wilcken et al., 2010). It is the use of bulb scales for treatment of asthma (Watt and Breyer- is a practice common in Sotho, Xhosa and Zulu culture carried out Brandwijk, 1962; De Beer and Van Wyk, 2011) and the use of bulb in a non-clinical setting by a traditional healer (an ‘Ngcibi’)with infusions for anxiety, depression and age-related dementia (Risa no formal medical training (De Smet, 1996; Foden, 2010; Wilcken et al., 2004a; Pedersen et al., 2008; Stafford et al., 2008; Neergaard et al., 2010). In the townships of Gauteng Province, 10% of males et al., 2009; Risa et al., 2011b). Interestingly, use of the plant for aged 14–24 years and 22% of those aged 19–29 years were respiratory ailments was shown to include both indigenous and reportedly circumcised, in 58–65% of cases by traditional circum- European respondents highlighting the cross-cultural significance cisers (Lagarde et al., 2003). In the same study, it was shown that of the plant. Apart from this Boophone haemanthoides, the only choice of providers depended on the affiliation to different ethnic other member of the genus Boophone is also utilised for respira- groups; for example, 86% of Xhosa participants were circumcised tory illnesses (De Beer and Van Wyk, 2011; Nair et al., 2012b). The by traditional healers compared with 37% of Tswana men. During widespread usage of Boophone disticha for neurological disorders the initiation period, Xhosa boys are covered from head to toe is well documented and further details of this will be provided in with sandstone which gives them the characteristic white colour Section 7 of this paper. In relation to specific diseases, Boophone and live together in a circumcision lodge under strict conditions disticha is applicable for treatment of various inflammatory con- of the lodge master who also instructs them in matters relating to ditions, cancer, malaria and tuberculosis (Table 1, entries 10–13). social conduct, duty, tradition and political obligation (Foden, Details of these functions, especially in cancer chemotherapy, will 2010). In Sotho tradition, initiates are given food mixed with also be provided in Section 7 of this paper. The three remaining bulbs of Boophone disticha together with other ingredients which categories pertain to psychoactive, divinatory and veterinary is thought to imbue them with the qualities of their ancestors as properties in which the plant finds common usage for therapeutic well as to make men out of them (De Smet, 1996). The signs of purposes. intoxication are regarded as a token that the spirit of manhood Of the manifold uses of Boophone disticha, its usage as an arrow had entered their bodies (De Smet, 1996). Furthermore, bulb and dart poison by San communities is most striking (Watt and infusions are used by Sotho and Xhosa boys as an outer dressing Breyer-Brandwijk, 1962; Verzár and Petri, 1987; Bisset, 1989; De for circumcision wounds (Watt and Breyer-Brandwijk, 1962; Smet, 1998). Amongst the varied purposes for which plant toxins Verzár and Petri, 1987; De Smet, 1996; Hutchings et al., 1996; have come to be used, their primary function as an auxiliary Viladomat et al., 1997; Grierson and Afolayan, 1999; Du Plooy weapon in the procurement of food has never been disputed et al., 2001; Van Wyk et al., 2008; Philander, 2011). More (Bisset, 1989; Philippe and Angenot, 2005). In addition, these recently, there have been concerted efforts to popularise the substances have been widely employed in tribal warfare, as items health benefits of medical male circumcision due to its protective in inter-tribal trade and as ingredients or sources of medicines effect in model studies of female-to-male transmission of HIV (Bisset, 1989; Philippe and Angenot, 2005). In present times it is (Wilcken et al., 2010). This not withstanding, traditional male believed that this practice still continues within some factions of circumcision is not without complications and numerous cases the San people scattered through parts of Namibia and Botswana are reported on an anual basis relating to excessive bleeding, (Bisset, 1989; De Smet, 1998). Moreover, it is known that the Khoi- infection, delayed wound healing and even death (Wilcken et al., San and certain Bantu tribes also once used Boophone disticha for 2010). Given these facts, the popularity of the practice on the similar purposes, but it is actively contended that the practice cultural landscape as well as the recent availability of male descended down from the San (Bisset, 1989; De Smet, 1998). The circumcision clinics nationwide, there has been ongoing debate Khoi-San used bulbs to poison arrows intended for shooting about the integration of this sector of TM into mainstream smaller types of game, and they appear to have had a profound healthcare in South Africa. J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 17

Table 1 Ethnopharmacological 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 and (a) Bulbs used by Sotho and Xhosa boys as an outer dressing for circumcision Hutchings et al. (1996), Nair et al. (2013), Viladomat et al. dietary wounds (1997), Watt and Breyer-Brandwijk (1962) (b) Outer layer of bulbs used to fashion head ring of Swati chief and headman Watt and Breyer-Brandwijk (1962) (c) Leaves are stripped to make fringes which are worn as decorative body Hutchings et al. (1996), Watt and Breyer-Brandwijk (1962) ornaments Bisset (1989), De Smet (1996, 1998), Neuwinger (1994), (d) Bulbs were used as an arrow and dart poison by San and Xhoi-San of the Cape Van Wyk et al. (2002, 2005), Watt and Breyer-Brandwijk (e) Bulbs known to have caused death by suicide in the OFS (1962) (f) Prior to tin utensils, scooped-out bulb was used by Sotho herd boys to warm Watt and Breyer-Brandwijk (1962) milk Watt and Breyer-Brandwijk (1962) (g) Zulu use bulb scales to plug ear lobes after piercing Hutchings et al. (1996) (h) Cape-Dutch settlers used a sleeping mattress ﬁlled with bulb scales to relieve Van Wyk et al. (2002), Watt and Breyer-Brandwijk (1962) insomnia Philander (2011) (i) Chewed dried leaves used to treat alcohol addiction in the Cape Hutchings et al. (1996) (j) Zulu take ﬂower infusions with porridge for dizziness

2. Well-being (a) Dry bulb scales taken for feeling of weakness Verzár and Petri (1987) (b) Fresh bulb water extract is drunk to increase sexual potency Verzár and Petri (1987) (c) Bulbs used to improve memory 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 to Cheesman et al. (2012) the Cape) for rashes, bruises, burns, cuts, wounds, boils and swelling. The treatment Grierson and Afolayan (1999) is thought to relieve pain as well as to draw out pus 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 Philander (2011) (b) Bulb decoctions are taken as an emetic and purgative Hutchings et al. (1996) (c) Bulbs used as enemas by the Sotho, Xhosa and Zulu Hutchings et al. (1996) (d) Fresh root and bulb decoction used for constipation 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 Hutchings et al. (1996) (b) Leaves used to stop bleeding Philander (2011), Viladomat et al. (1997)

7. Respiratory (a) Papery bulb scales used for asthma by Zulu and San De Beer and Van Wyk (2011), Van Wyk et al. (2008) and (b) Bulbs used for dyspnoea Watt and Breyer-Brandwijk (1962) (c) Zulu women roll snuff on a piece of dried bulb scale to improve the efﬁcay of Watt and Breyer-Brandwijk (1962) the snuff Watt and Breyer-Brandwijk (1962)

8. Muscular (a) Bulb decoctions are applied for muscle pain and stiffness Viladomat et al. (1997), Watt and Breyer-Brandwijk (1962) (b) Oral bulb decoction given to Zulu patients suffering from ‘inkwatshu’, Hutchings et al. (1996), Viladomat et al. (1997) characterised by cramp-like pain in the calf muscles and tightness in the ﬁngers and toes

9. Neurological (a) Roots are burned, ground and the powder applied to the area experiencing Verzár and Petri (1987) paralysis Watt and Breyer-Brandwijk (1962) (b) An old European remedy for hysteria from the Touws River area involved Nielsen et al. (2004), Watt and Breyer-Brandwijk (1962) sleeping on a mattress ﬁlled with bulb scales Nyazema (1986), Pedersen et al. (2008), Sobiecki (2008) (c) Bulbs from the Umtentweni area of the KwaZulu-Natal south coast are used in Hutchings and Van Staden (1994) the treatment of ‘fufunya’ (a type of hysteria) Neergaard et al. (2009), Pedersen et al. (2008), Sandager (d) Bulb decoctions administered to pshychotic patients et al. (2005), Stafford et al. (2008) (e) Bulb extracts taken orally by Sotho, Xhosa and Zulu for stress-related ailments Risa et al. (2004a, 2011b), Stafford et al. (2008) (f) Bulb infusions drunk to relieve various mental illnesses, including anxiety and depression (g) Bulb extracts used for age-related dementia

10. Inﬂammatory (a) Milk in which bulb scales have been macerated was used by Europeans in the Hutchings et al. (1996), Watt and Breyer-Brandwijk (1962) conditions Lydenburg district to treat various inﬂammatory conditions Botha et al. (2005), Van Wyk et al. (2002, 2005), Watt and (b) Moistened bulb scales were also known to be used by early settlers for Breyer-Brandwijk (1962) rheumatic pain Verzár and Petri (1987), Watt and Breyer-Brandwijk (1962) (c) Bulbs are applied for eye conditions as well as healing of infected scars Hutchings et al. (1996), Viladomat et al. (1997) (d) Weak bulb decoctions administered orally or via enema to Zulu adults for Watt and Breyer-Brandwijk (1962) stomache, headache, chest and bladder pain Mabona and Van Vuuren (2013), Watt and Breyer- (e) The Manyika apply bulb scales locally for relief from urticaria Brandwijk (1962) (f) Dried leaves moistened with milk or oil is used to treat skin diseases, varicose Hutchings et al. (1996), Viladomat et al. (1997), Watt and ulcers and phlebitis Breyer-Brandwijk (1962) (g) Fresh leaves applied by Zulu to check bleeding 18 J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26

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 to Watt and Breyer-Brandwijk (1962) treat tuberculosis

14. Psychoactive (a) Weak bulb dococtions used as a sedative to calm psychotic patients Pedersen et al. (2008), Sobiecki (2008) properties (b) Bulb concoctions taken orally have caused sedation, analgesia, visual Du Plooy et al. (2001), Neergaard et al. (2009) hallucinations, unconsciousness, irrational behaviour, talkativeness as well as Van Wyk et al. (2002) coma Hutchings et al. (1996), Sandager et al. (2005) (c) Induce hallucinations for divinatory purposes Neergaard et al. (2009), Stafford et al. (2008) (d) Used as a narcotic substance (e) Bulbs known to induce trance

15. Divinatory (a) The Manyika grow the plant outside their huts as a charm to ward off evil Watt and Breyer-Brandwijk (1962) purposes dreams, to bring goodluck and rain as well as to keep away the ‘mudzimu’ Neergaard et al. (2009), Stafford et al. (2008), (wandering spirit) after a death Du Plooy et al. (2001), Gadaga et al. (2010), Hutchings et al. (b) Psychoactive properties of the bulbs used to induce trance (1996), Philander (2011), Van Wyk et al. (2002) (c) Used in various divination rituals such as arousing the ancestral spirits and Van Wyk (2008) curing demonic possession (d) Thought to symbolise eternal life

16. Veterinary uses (a) The Xhosa use the bulbs as a remedy for redwater in cattle Watt and Breyer-Brandwijk (1962) (b) Cattle are known to browse the plant with impunity Verzár and Petri (1987) (c) It has been suggested that vultures and other carrion birds eat the plant to Watt and Breyer-Brandwijk (1962) prevent harm from ingestion of putrid ﬂesh or to sharpen vision

In addition to the widespread cultural significance of Boophone characteristic (Viladomat et al., 1997; Bastida et al., 2006; Nair disticha in areas as diverse as ‘arrow and dart poisons’ as well as et al., 2013). These now collectively represent a large and expand- ‘initiation and circumcision rituals’, the plant is most widely ing group of alkaloids with a distinct array of structural diversity known for several of its neurological functions such as treatment in which galanthamine 1, lycorine 2 and crinine 3 feature as the of hysteria, stress-related ailments, psychosis, anxiety and depres- three major structural-types (Scheme 1)(Viladomat et al., 1997; sion as well as age-related dementia (Watt and Breyer-Brandwijk, Bastida et al., 2006; Jin, 2011; Nair et al., 2013). By contrast, 1962; Hutchings et al., 1996; Pedersen et al., 2008; Stafford et al., homolycorine 4, tazettine 5 and montanine 6 make up the minor 2008)(Table 1). Interestingly, a global systematic survey of 143 series of compounds while trisphaeridine 7 and ismine 8 repre- plants used to treat diseases of the nervous system showed that sent some of the less-conspicuous members (Viladomat et al., seven representatives were from the family Amaryllidaceae, 1997; Bastida et al., 2006; Jin, 2011; Nair et al., 2013). All these including Ammocharis coranica, Boophone disticha, Scadoxus puni- structures share the same biosynthetic precursor (norbelladine 9) ceus, Galanthus nivalis, Lycoris radiata, Narcissus jonquilla and from which they are derived via different sequences of phenolic Narcissus pseeudonarcissus, of which the first three are African in oxidative coupling (Viladomat et al., 1997; Bastida et al., 2006; Jin, origin (Gomes et al., 2009). Furthermore, based on these findings 2011; Nair et al., 2013). To date over 500 of these alkaloids have all plants covered in the survey were suggested as being possible been identified in Amaryllid species across the globe (Viladomat complementary or alternative therapies for neurological and et al., 1997; Bastida et al., 2006; Jin, 2011; Nair et al., 2013). The neurodegenerative disorders (Gomes et al., 2009). An often over- South African contingent presently stands at 193 alkaloids, repre- looked area in this sector is the psychoactive properties of senting roughly 39% of the global complement of structures, Boophone disticha leading to it being commonly classified as an which have been isolated from 88 species spanning 14 genera ‘African hallucinogen’ (De Smet, 1996; Sobiecki, 2008; Foden and three tribes (Viladomat et al., 1997; Nair et al., 2013). 2010). In the practice of trance, the Khoi-San believed that the Furthermore, this number is dividable into the different groups plant could serve as a doorway to the spirit of the dead. Sangomas of compounds characteristic of the Amaryllidaceae, including 84 are also known to take the plant to psychoanalyse their patients, a crinanes, 40 lycoranes, 7 galanthamine compounds, 9 tazettine process they call ‘bioscope’ hence the common name for Boophone representatives, 27 homolycorine compounds, 10 montanine ana- disticha (‘bioscope plant’)(De Smet, 1996; Sobiecki, 2008; Foden logues and 16 miscellaneous alkaloids (Viladomat et al., 1997; Nair 2010). et al., 2013). Although crinane alkaloids (such as crinine 3) with 84 representatives are the most populous of the South African Amaryllidaceae, lycorine 2 is by far the most common compound 6. Phytochemistry having been isolated on 38 separate occasions i.e. it is present in 43% of plants examined (Viladomat et al., 1997; Nair et al., 2013). 6.1. Alkaloids of the South African Amaryllidaceae Given this statistic alone coupled to the significant array of biological properties demonstrated by lycorine, it is not surprising Although several classes of secondary metabolites are known that the Amaryllidaceae continues to attract widespread interest to occur in the Amaryllidaceae, such as chalcones, flavonoids, with the status as a biologically privileged plant family. Further- lectins, lignans, peptides and terpenoids, it is the isoquinoline more, the commercial success of galanthamine 1, the clinical alkaloids which have endowed the family with a unique chemical potential of compounds such as pancratistatin 10 as well as the J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 19 natural abundance of the Amaryllidaceae in South Africa, where the structural features of these alkaloids, crinane compounds less than a third of the species have been studied, have together possess a basic phenanthridine nucleus with varying degrees of galvanised the plant family as a truly viable vehicle for drug oxidation in ring-A, but usually with a methylenedioxy bridge discovery. across C-8 and C-9 (as in crinine 3 and buphanisine 15)(Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013). In addition, 6.2. Alkaloids from Boophone disticha methoxy substitution at C-7 is common for many analogues across the series, such as buphanidrine 13 and undulatine 14 (Viladomat Although various compounds such as furfuraldehyde, acetova- et al., 1997; Bastida et al., 2006; Nair et al., 2013). Substitution in nillone, chelidonic acid, copper, laevulose, pentatriacontane, a ring-B is infrequent, but when it is present it is normally at C-6 (as phytosterol, ipuranol and fatty acids were initially reported to be in 6-hydroxycrinamine 19) or C-4a (as in crinamabine 20) present in bulbs of Boophone disticha (Tutin, 1911a), its alkaloid (Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013). In constituents have subsequently gained prominence due to their addition, protonated salts bearing chloride counter anions, as well important biological properties. Given the long history of usage of as N-oxide derivatives of several crinane compounds have been Boophone disticha in TM in South Africa, it is not surprising that the identified (Viladomat et al., 1997; Bastida et al., 2006; Nair et al., plant was amongst the first of the Amaryllidaceae species globally 2013). In relation to ring-C, oxygen-related substituents are known to be examined for alkaloid constituents (Tutin, 1911a,b; Lewin, to occur at all exchangeable positions, including C-1, C-2, C-3 and 1912a,b; Tutin, 1913). Furthermore, the plant serves as a flagship C-4, and a C-1 to C-2 double bond is common for many derivatives for phytochemical endeavours emanating out of the southern (Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013). African region. Given the complexity of these structures, the early A distinguishing feature of crinane alkaloids is the presence of the years of chemical studies on Boophone disticha was understand- ethano-bridge straddling N-5 and C-10b which may be either α-or ably arduous. The first phytochemical investigation of the plant β-orientated, leading to the absolute stereochemical configuration was that performed by Tutin (1911a,b) who described the presence at C-4a. In the case of lycorine 2, the C-terminus of the ethano- of lycorine 2 together with three other unknown alkaloids. bridge is at C-4 and C-10b reverts to a tertiary carbon centre Following this Lewin (1912a,b) isolated a compound loosely (Viladomat et al., 1997; Bastida et al., 2006; Nair et al., 2013). Of referred to as “haemanthine”, which Tutin (1913) maintained the compounds isolated from Boophone disticha, distichamine 16 is was in fact a mixture. However, evidence in support of Lewin's most noteworthy for its unique ring-C substitution pattern stance for the chemical individuality of haemanthine was provided (Viladomat et al., 1997; Nair et al., 2012a, 2013). In its structure, by Cooke and Warren (1953) based on elemental analysis, melting oxidation has occurred at C-1 in accommodating the keto group points and semi-synthetic derivatisation. By contrast, support for and the double bond has shifted to the C-2/C-3 position with the stance taken by Tutin is reflected in studies carried out several concomitant vinylization of the C-3 methoxy group (Viladomat decades later which revealed that “haemanthine” was a compo- et al., 1997; Nair et al., 2012b, 2013). Such a substitution pattern nent mixture of buphanamine 11 or nerbowdine 12 (Bates et al., has never been found for any other alkaloid within the crinane 1957; Goosen and Warren, 1957; Fales and Wildman, 1961). series (Viladomat et al., 1997; Nair et al., 2012b, 2013). Further- Following on these early investigations, Bates et al. (1957) isolated more, distichamine 16 has never been found outside Boophone and lycorine 2, buphanamine 11 and buphanidrine 13 from bulbs of thus reserves the privilege as a distinctive chemotaxonomic Boophone disticha collected at various locations in South Africa, marker for the genus (Viladomat et al., 1997; Nair et al., 2012b, including Bloemfontein, Grahamstown, Pietermaritzburg and Pre- 2013). toria. However, to date the most comprehensive phytochemical study carried out on the plant is that of Hauth and Stauffacher (1961) who described the presence of 11 alkaloids in ethanolic 7. Pharmacology bulb extracts, including buphanidrine 13 (19.4%), undulatine 14 (18.6%), buphanisine 15 (16.9%), buphanamine 11 (14.1%), nerbow- 7.1. Antimicrobial activity dine 12 (11.1%), crinine 3 (7.2%), distichamine 16 (5.4%), crinamidine 17 (1.2%), acetylnerbowdine 18 (0.6%), lycorine 2 (0.4%) and Given that Boophone disticha is the most common representa- buphacetine (0.3%) (where percentage values are expressed as tive of the South African Amaryllidaceae used to treat wounds and relative contribution to total alkaloids). Whereas majority of these infections (Hutchings et al., 1996; Rabe and Van Staden, 1997; compounds have been subsequently verified through physical and Grierson and Afolayan, 1999; Shale et al., 1999), it is not surprising spectroscopic means (Viladomat et al., 1997; Nair et al., 2013), the that some studies have been directed towards gauging its efficacy identity of buphacetine remains a mystery to this day. Interest- against bacterial pathogenesis (Heyman et al., 2009; Cheesman ingly, buphanidrine, buphanisine, crinine and distichamine were et al., 2012). An ethanolic bulb extract of Boophone disticha was also found in Boophone haemanthoides, but at percentages of active against methicillin-sensitive Staphylococcus aureus with an 46.9%, 23.9%, 7.3% and 21.9%, respectively (Nair et al., 2012b). MIC of 7.25 mg/mL (Heyman et al., 2009). Interestingly, the same Further studies of Boophone disticha confirmed the presence of extract was shown to be potentially fatal to the pathogen as a buphanamine, buphanidrine, buphanisine, crinine and disticha- minimum bactericidal concentration (MBC) was established at mine (Sandager et al., 2005; Neergaard et al., 2009; Cheesman 12.5 mg/mL (Heyman et al., 2009). Further work on Boophone et al., 2012). The most recent phytochemical investigation of disticha by Cheesman et al. (2012) initially showed bulb extracts to Boophone disticha led to the isolation of 6-hydroxycrinamine 19 be active against two Gram-positive (Bacillus subtilis and Staphy- from a methanolic bulb extract at a concentration of 0.01% dry lococcus aureus) and two Gram-negative (Escherichia coli and weight (Adewusi et al., 2012). It is clear from these collective Klebsiella pneumoniae) strains that made up the microdilution

ﬁndings that, with the exception of lycorine 2, the alkaloids bacterial screen, with IC50s in the range 0.13–0.25 mg/mL. Led by produced by Boophone disticha are all the crinane series of these promising activities, bioassay-guided fractionation then Amaryllidaceae alkaloids. Furthermore, 6-hydroxycrinamine 19 resulted in the isolation of two active crinane alkaloid constitu- was the only compound identiﬁed belonging to the α-crinane ents: buphanidrine 13 and distichamine 16 (Scheme 1)(Cheesman sub-series (Adewusi et al., 2012), the others being characteristic of et al., 2012). In the repeat screen, best activities were seen against β-crinane alkaloids (Hauth and Stauffacher, 1961; Sandager et al., Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae

2005; Neergaard et al., 2009; Cheesman et al., 2012). In terms of with both compounds exhibiting IC50s of 0.063 mg/mL against all 20 J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 three pathogens (correspondent with micromolar values of 200 control (10 mg/kg) (Citoglu et al., 1998). In addition, crinine 3 and and 191.5 for buphanidrine 13 and distichamine 16, respectively) crinamidine 17, both known alkaloid constituents of Boophone (Cheesman et al., 2012). Both compounds were two-fold less active disticha (Hauth and Stauffacher, 1961), exhibited mild anti- against Bacillus subtilis (IC50 0.13 mg/mL) as also seen in the screen inflammatory activity as determined through COX inhibition against the crude extract (Cheesman et al., 2012). These results, (Elgorashi et al., 2003). To this extent, crinine 3 and crinamidine 17 – though not as potent as the neomycin standard (IC50s of 0.8 10 at a concentration of 500 μM exhibited respective inhibitory activities 3–1.6 10–3 μg/mL) (Cheesman et al., 2012), are nevertheless of16%and10%againstCOX-1(Elgorashi et al., 2003). Interestingly, consistent with previous observations on the low-to-mild anti- this activity was seen to be selective as COX-2 remained unaffected bacterial activities of Amaryllidaceae alkaloids (Viladomat et al., by similar treatments using either compound (Elgorashi et al., 2003). 1997; Elgorashi et al., 2003; Bastida et al., 2006; Nair et al., 2013). Amongst the South African Amaryllidaceae only Boophone disticha 7.3. Effects on the central nervous system is known in TM for treating tuberculosis (Watt and Breyer- Brandwijk, 1962), although no pharmacological studies have been 7.3.1. Hallucinogenic effects carried out to verify this property. The use of plants as a source of chemicals with CNS-activating Information pertaining to the usage of South African Amarylli- properties is common on the South African cultural landscape. As daceae plants for fungal infections is sparse in the literature. such, over 300 plant taxa have been identified within this context Although, Boophone disticha is not indicated for such purposes, of TM (Stafford et al., 2008). At least three Amaryllid plants are several others, including Amaryllis belladonna, Crinum macowanii known from local tradition to be exploited for hallucinatory and Crinum moorei, have shown antifungal or antiyeast activity purposes (De Smet, 1996; Viladomat et al., 1997; Stafford et al., (Viladomat et al., 1997). In terms of activities of single compound 2008). Pancratium tenuifolium is used by the San of the Kalahari to isolates, lycorine 2 and vittatine 21 were reported with significant induce visual hallucinations while Brunsvigia radulosa is well- activity against Candida albicans (MICs of 39 and 31 μg/mL, known from San culture as a psychoactive plant (Viladomat respectively) (Evidente et al., 2004). In relation to antiviral activity, et al., 1997). In addition, Boophone disticha is recognised as a again the literature is devoid of any mention of the traditional hallucinogenic plant in both San and Sotho traditions (Viladomat usage of Boophone disticha for such purposes. However, the plant et al., 1997; De Smet, 1996; Neuwinger, 1994; Neuwinger and has been suggested to be effective against different rhinovirus Mebs, 1997; Sobiecki, 2002, 2008; Stafford et al., 2008). It has been strains (Viladomat et al., 1997). In regards to activities of individual suggested that its alkaloid constituents, either as single com- compounds, lycorine 2 has been shown to be active against a pounds or additively, are largely responsible for these effects of number of viruses (Bastida et al., 2006), including poliomyelitis the Amaryllidaceae (Viladomat et al., 1997; Bastida et al., 2006; virus at concentrations as low as 1 μg/mL (Ieven et al., 1982). Nair et al., 2013). Of the Amaryllidaceae alkaloids, galanthamine 1 Structure–activity relationship studies involving Herpes simplex (Scheme 1) is best known for its narcotic effects such as induction virus showed that lycorine exerted its antiviral effect by blocking of “lucid dream” (LD) and “out of body experience” (OBE) as well DNA polymerase activity (Ieven et al., 1983). Other compounds of as its application as a nootropic agent (Harvey, 1995; Heinrich and the family which have demonstrated antiviral activity in model Teoh, 2004). Of the compounds isolated from Boophone disticha, studies include homolycorine 4, tazettine 5, narciclasine 22 and buphanidrine 13 is known to have hyoscine-like activity i.e. it haemanthamine 23 (Scheme 1)(Bastida et al., 2006). In terms of exhibits muscarinic antagonist effects (Viladomat et al., 1997; Nair antiparasitic activity of South African Amaryllidaceae plants, only et al., 2013). Furthermore, since the identity of buphacetine has Amaryllis belladonna is known to be active against Plasmodium never been clarified, it could be speculated that this alkaloid may gallinaceum (Viladomat et al., 1997). Amongst all these plants, only be responsible for the hallucinogenic properties of the plant. In Boophone disticha is known to be used traditionally to treat addition, it is possible that all (or some) the alkaloids known from malaria (Watt and Breyer-Brandwijk, 1962), although no studies Boophone disticha could together function in a synergistic manner have been directed towards gauging the efficacy of plant extracts to produce the observed hallucinogenic effects. Symptoms of these against the malarial parasite. Of the various groups of Amarylli- effects include unconsciousness, dilated pupils, tachycardia, raised daceae alkaloids screened against the malarial parasite, lycorine 2 blood pressure, slightly raised temperature, laboured respiration, was the most potent against both chloroquine-sensitive (D10) and psychosis, drunkenness and visual disturbances as reported by chloroquine-resistant (FAC8) strains of Plasmodium falciparum Laing (1979) subsequent to treating patients presenting with with IC s of 0.6 and 0.7 μg/mL, respectively (Campbell et al. 50 Boophone disticha intoxication. 1998, 2000; Şener et al., 2003).

7.2. Anti-inflammatory activity 7.3.2. Mental disorders 7.3.2.1. Anxiety and stress. In addition to the psychoactive properties ‘Inflammation-related conditions’, ‘wounds and infections’ and of Boophone disticha,theplantisalsousedtotreatmentaldisorders ‘ailments pertaining to the CNS’ together represent the top three such as anxiety, depression, epilepsy and age-related dementia categories under which Boophone disticha finds most extensive use in (Stafford et al., 2008; Gomes et al., 2009). Anxiety disorders are the TM in South Africa (Watt and Breyer-Brandwijk, 1962; Verzár and most common psychiatric illnesses encountered in clinical practice Petri, 1987; De Smet, 1996; Hutchings et al., 1996; Viladomat et al., (Pote et al., 2013). Often they are chronic conditions associated 1997; Grierson and Afolayan, 1999; Du Plooy et al., 2001; Van Wyk with considerable cardiovascular-related morbidity and mortality et al., 2008; Philander, 2011). Verification of the usage of the plant for (Schwartz and Nihalani, 2006). Although information pertaining to inflammation-related conditions came from cyclo-oxygenase (COX) the pharmacological basis to the usage of Boophone disticha for inhibition studies in which ethanolic bulb extracts of Boophone anxiety and stress is largely absent from the literature, a recent study disticha exhibited 55% inhibition against COX-1 (Jäger et al., 1996). employed a mouse model to validate this claim (Pote et al., 2013). In terms of the activities of single compound extractives, lycorine 2 is Maternal separation is a common animal model used to examine the known to possess anti-inflammatory properties as shown through long-term effects of early life experience on subsequent behaviour in the carrageenan-induced paw oedema test in mice (Citoglu et al., adulthood and the development of psychiatric disorders such as 1998). At a dosage of 100 mg/kg lycorine induced 20.1% inhibition of anxiety and depression (Pryce and Feldon, 2003). Furthermore, oedema in the test subjects compared to 29.1% by the indomethacin repeated early maternal separation stress is known to produce an J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 21 altered effect in cardiovascular response in such models of study 65 and one in five over the age of 80, with an estimated annual (Pryce and Feldon, 2003). In this regard, ethanolic extracts of cost of over $600 billion (McNulty et al., 2010; Nair and Van Boophone disticha were used to gauge the effects on blood pressure Staden, 2012). By contrast, CNS disorders associated with old age and heart rate in adult BALB/c mice subjected to repeated early such as AD, amyotrophic lateral sclerosis (ALS) and Parkinson's maternal separation stress (Pote et al., 2013). The results revealed disease (PD) are not a major concern for the healthcare sectors of that maternally separated mice treated with a low dose (10 mg/kg/ developing nations, which has been attributed to a number of BW/day) of Boophone disticha had the lowest systolic blood pressure, factors, including genetic, dietary, lifestyle and environmental whereas those treated with a high dose (40 mg/kg/BW/day) exhibited measures entrenched in these societies (Stafford et al., 2008). the lowest diastolic blood pressure and mean arterial pressure Given this rising health concern for developed nations, the search following acute stress in adulthood compared to the control and and development of chemical targets into suitable therapies diazepam treated animals (Pote et al., 2013). These findings suggest against this complex of neuronal diseases is at a premium that postnatal stress can induce short-term changes in the sensitivity (Heinrich and Teoh, 2004; Houghton et al., 2006; Nair and Van of the cardiovascular system to subsequent stress which can be Staden, 2012). In this regard, plants provide a substantial resource reduced by treatment with Boophone disticha. base for such efforts in drug discovery, some of which have already delivered effective candidates such as rivastigmine, huperzine A 7.3.2.2. Depression. Several antidepressant drugs available in clinical and galanthamine (also referred to as galantamine) to the clinical practice, such as citalopram, fluoxetine and paroxetine, exert their market (Heinrich and Teoh, 2004; Houghton et al., 2006). These fi effects through selective inhibition of serotonin reuptake and are compounds are highly ef cient at AChE inhibition (IC50 for μ fi pharmaceutically classed as selective serotonin reuptake inhibitors galanthamine is 1 M), a process thought to be signi cant in the fi (SSRIs) (Nielsen et al., 2004). Mechanistically, such compounds are pathophysiology of AD, which ts the premise that Alzheimer's fi known to bind to the SSRI binding site on the neuronal serotonin disease is associated with cholinergic insuf ciency (Harvey, 1995; transporter thus inhibiting the transport of serotonin from the Greig et al., 2004; Heinrich and Teoh, 2004; Houghton et al., 2006). synaptic gap back to the neuron (Stahl, 1998). In the competitive Given the commercial success of galanthamine 1 (Scheme 1), it is binding assay with 3H-labelled citalopram, aqueous leaf and bulb clear that the plant family Amaryllidaceae invokes widespread extracts of Boophone disticha resulted in the displacement of more interest as a resourceful platform for discovery in this area (Lopez than 50% of the transport protein bound [3H]citalopram at tested et al., 2002; Elgorashi et al., 2004, 2006a; McNulty et al., 2010; concentrations of 5, 1 and 0.1 mg/mL (Nielsen et al., 2004). Buoyed by Monton et al., 2010; Nair et al., 2011; Nair and Van Staden, 2012). these findings, bioassay-guided fractionation then led to the isolation Such endeavours have highlighted the potency of other alkaloids μ of the alkaloids buphanamine 11, buphanidrine 13, buphanisine 15 such as sanguinine 24 (IC50 0.1 M) and 1-O-acetyllycorine 25 (IC50 μ and distichamine 16 (Scheme 1)frombulbsofBoophone disticha, 0.96 M) (Scheme 1) as AChE inhibitors with genuine potential for clinical advancement (Lopez et al., 2002; Elgorashi et al., 2004, which exhibited respective IC50 values of 55, 62, 199 and 65 μMinthe serotonin binding assay (Neergaard et al., 2009). In addition, 2006a; McNulty et al., 2010; Monton et al., 2010; Nair et al., 2011; buphanamine 11 and buphanidrine 13 demonstrated affinities to the Nair and Van Staden, 2012). The activity of sanguinine is not unexpected given its close structural similarity to the standard AChE serotonin transporter in rat brain with IC50sof1799and274μM, respectively (Sandager et al., 2005). Apart from this, Pedersen et al. inhibitor galanthamine. However, its potency over galanthamine (2008) employed a rat model for depression to show that Boophone must reside with its ring-A phenolic hydroxyl group (which in the disticha extracts inhibited the serotonin transporter (SERT), case of galanthamine is substituted by a methoxyl group). The role of noradrenalin transporter (NAT) as well as the dopamine transporter the methoxyl group in galanthamine has been shown to be sig- fi (DAT), all of which are believed to be involved in the pathophysiology ni cant as it occupies the acyl-binding pocket, contacting both Phe- 288 and Phe-290 amino acid residues within the active site, and is of depression, with IC50s of 423.8, 77.3 and 93.5 μMrespectively. Furthermore, antidepressant-like effects were observed for extracts of therefore an essential structural feature of this AChE inhibitory Boophone disticha in the tail suspension test (TST) and the forced swim pharmacophore (Sussman et al., 1991; Greenblatt et al., 1999). The test (FST) in both rats and mice (Pedersen et al., 2008). activity of sanguinine 24 over galanthamine 1 suggests that a small polar group in the C-9 region of the molecule, capable of hydrogen bond donor and acceptor functions (reminiscent of a phenolic fi 7.3.2.3. Epilepsy. Epilepsy is a neurological condition typi ed by hydroxyl), is better accommodated at the active site. repeated and unprovoked seizures often accompanied by severe The potency of 1-O-acetyllycorine 25 at AChE inhibition is due convulsions (Risa et al., 2004a). Benzodiazepines are a common class chiefly to its structural proximity to galanthamine and the possibility of anti-convulsing agents which bind to the GABAA benzodiazepine of overlap along significant areas of their respective pharmacophores fi receptor complex where they enhance the af nity for the inhibitory (Elgorashi et al., 2006a; Nair et al., 2011). Quantitative structure– γ neurotransmitter -aminobutyric acid (GABA) (Risa et al., 2004a). activity relationship (QSAR) studies involving superposition of mini- fl AGABAstimulusontheGABAA-receptor causes an in ux of chloride mum energy conformations showed partial overlap for the methoxyl ions into the cell producing hyperpolarisation of the membrane, of galanthamine and the methylenedioxy of 1-O-acetyllycorine fi making it more dif cult to generate action potential, resulting in cell (Elgorashi et al., 2006a; Nair et al., 2011). Furthermore, the acetate arrest and consequent anti-convulsant activity (Risa et al., 2004a). and nitrogen atom of 1-O-acetyllycorine superimposed on the Although Boophone disticha isknownintheTMapproachtoepilepsy hydroxyl group and the nitrogen atom of galanthamine, respectively (Stafford et al., 2008), no pharmacological investigation has sought to (Elgorashi et al., 2006a; Nair et al., 2011), making it possible for the fl clarify this usage. By contrast, the Amaryllid Brunsvigia grandi ora acetyl group to participate in hydrogen bonding in a manner similar fi exhibited high binding af nities (up to 100%) for the GABAA- to that demonstrated for the hydroxyl of galanthamine (Greenblatt fl benzodiazepine receptor in competition with umazenil (Risa et al., et al., 1999). In addition, the C-4/C-4a double bond in galanthamine 2004a). Furthermore, none of the components of a structurally was seen to have ππ interactions with Trp-84 (Greenblatt et al., diverse Amaryllidaceae alkaloid mini-library were responsive to 1999),andsincethelycorinedoublebondisinaproximallysimilar GABAA-benzodiazapine receptor binding (Elgorashi et al., 2006b). position it may be able to elicit a similar response at the active site (Elgorashi et al., 2006a; Nair et al., 2011). 7.3.2.4. Age-related dementia. The impact of age-related dementia Boophone disticha is one of the seven different South African on western medicine is staggering, affecting one in 20 people over Amaryllidaceae plants shown to be effective in the treatment of 22 J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 age-related dementia and memory loss in TM (Stafford et al., embracive of the anti-cancer properties of the country's floral 2008). As such, verification of this property was arrived at via biodiversity. To this extent, 7500 randomly selected plant extracts AChE inhibition assays by which aqueous and ethanolic bulb (representing 700 species) were tested in the three cell line pre- extracts exhibited inhibitory activities of 37% and 30%, respectively screen using MCF7, TK10 and UACC62 cells at a single dose of (Risa et al., 2004b). Of the 12 compounds isolated from Boophone 100 μg/mL (Fouche et al., 2006, 2008). Out of these, a total of 950 disticha, lycorine 2, 6-hydroxycrinamine 19 and crinine 3 exhibited extracts exhibited growth inhibitions of 75% or higher against two AChE inhibitory activities of 29.3, 445 and 461 μM respectively or more of the cancer cells screened (Fouche et al., 2006, 2008). (Elgorashi et al., 2004; Adewusi et al., 2012; Elisha et al., 2013), The traditional use of Amaryllidaceae plants for cancer is which consolidate the traditional usage of the plant for these known from all three geographical regions of its prominence, conditions (Stafford et al., 2008; Gomes et al., 2009). including Andean South America, the Mediterranean and South In addition to AChE inhibitors, monoamine oxidase (MAO) Africa (Pettit et al., 1984; Nair et al., 1998; Botha et al., 2005; inhibitors are also known to be effective in the treatment of AD Kornienko and Evidente, 2008; Caamal-Fuentes et al., 2011). and PD (Stafford et al., 2007). MAO enzymes are present in the Amaryllis belladonna, Boophone disticha and Crinum delagoense outer mitochondrial membrane of neuronal and non-neuronal are three of the local Amaryllid plants known to be used in TM cells and are responsible for the oxidative deamination of endo- for treating this disease (Botha et al., 2005; Nair et al., 1998; Pettit genous as well as xenobiotic amines (Yamada and Yasuhara, 2004). et al., 1984). Apart from its clinical relevance to MND, the plant The two known isoforms MAO-A and MAO-B are differentiated by family Amaryllidaceae is most widely known in pharmacology for substrate preference, inhibitor specificity and tissue distribution its anti-cancer properties (Kornienko and Evidente, 2008; (Yamada and Yasuhara, 2004). MAO-A preferentially deaminates Evidente and Kornienko, 2009; Nair et al., 2012a). Furthermore, serotonin, adrenaline and noradrenaline and makes up around 75% its alkaloid constituents are known to be responsible for these of MAO in human brain tissue, while MAO-B is specific for effects as shown via both in vitro and in vivo models of study substrates such as dopamine, β-phenylethylamine and benzyla- (Kornienko and Evidente, 2008; Evidente and Kornienko, 2009; mine (Yamada and Yasuhara, 2004). In relation to this, Stafford Nair et al., 2012a). Although cytotoxic activities are detectable et al. (2007) screened a collection of 20 different plant taxa known across all series of alkaloids of the Amaryllidaceae, the crinane and to be used in South African TM for age-related dementia for MAO lycorine alkaloids are the most common targets of choice in such activity. In the study including the two Amaryllids Boophone studies due to their potency (Evidente and Kornienko, 2009; disticha and Scadoxus puniceus, only the latter was active in non- Lamoral-Theys et al., 2010; Nair et al., 2012a). As such, over 100 selective MAO inhibition (IC50 406 μg/mL) and selective MAO-B different crinane and lycorine structures have been evaluated for inhibition (IC50 344 μg/mL) (Stafford et al., 2007). This result anti-tumour activity (Evidente and Kornienko, 2009; Lamoral- highlights the utility of Boophone disticha as a source of selective Theys et al., 2010; Nair et al., 2012a). Of the 12 alkaloids reported chemotherapeutics against motor neuron disease, given its activity from Boophone disticha (Hauth and Stauffacher, 1961; Sandager in AChE inhibition. et al., 2005; Neergaard et al., 2009; Adewusi et al., 2012; Cheesman et al., 2012), all with the exception of nerbowdine 12, 7.4. Anticancer activity acetylnerbowdine 18 and buphacetine have been subjected to cytotoxicity screening against various cancer cells (Evidente and Cancer is a leading cause of death worldwide, accounting for Kornienko, 2009; Lamoral-Theys et al., 2010; Nair et al., 2012a). In 7.6 million deaths (around 13% of all deaths) in 2008 (WHO, 2008). this regard, studies on lycorine 2 (Scheme 1) are significant in that During this period 669,000 deaths as a result of cancer, including it was amongst the first of the Amaryllidaceae alkaloids to exhibit 349,000 in men and 320,000 in women, were recorded in North cytotoxic activity as shown by its inhibitory effects towards cell America (WHO, 2008). Such statistics for the African continent are division and cell elongation, as well as protein synthesis in more difficult to come by since a very small proportion of the total eukaryotic cells (De Leo et al., 1973; Jimenez et al., 1976). population is covered by medically certified causes of death or Furthermore, both in vitro and in vivo models of leukaemia (HL- population-based cancer registries (WHO, 2008). However, con- 60) cells support the potency of lycorine as an antiproliferative servative estimates have put this number around 518,000 for agent (Liu et al., 2004, 2007). Notably, in some cell lines these deaths from cancer in the African region for 2008 (WHO, 2008). effects were manifested via the apoptotic mechanistic pathway Of greater concern is that global deaths from cancer are set to rise (Liu et al., 2007; Li et al., 2007; McNulty et al., 2009a). By contrast, sharply with 17 million cases projected for 2030 (WHO, 2008). In antiproliferative effects by lycorine 2 were also exerted in North America, lung, stomach, liver, colon and breast cancers apoptosis-resistant cell lines such as OE21 oesophageal cancer together cause the most number of deaths from cancer each year, and SKMEL-28 melanoma cells (Van Goietsenoven et al., 2010). In of which lung cancer is forecast as the leading cause for both terms of potency, IC50s as low as 0.5 μM were observed for lycorine males and females in 2013 (Cancer Facts & Figures, 2013). Given 2 against various leukaemia and lymphoma cell lines, while for these facts and their increasing burden on the healthcare sector, carcinomas, multiple myeloma and melanoma cells IC50swerein the search for effective new therapies is an ongoing concern for the range 3–10 μM(Lamoral-Theys et al., 2010). In vivo, HL-60 health organisations worldwide. Present therapies include che- leukaemia-bearing mice exhibited a T/C index of 134% at a lycorine motherapy, immunotherapy, monoclonal antibody therapy, radia- treatment dosage of 10 mg/kg (Lamoral-Theys et al., 2010). Simi- tion therapy and surgery. In this regard, plants provide a vast larly, crinane alkaloids of the Amaryllidaceae have come into focus resource base for chemotherapeutic based anti-cancer drug dis- since the identification of haemanthamine 23 as a cytotoxic agent covery (Graham et al., 2000; Balunas and Kinghorn, 2005; Cragg over 30 years ago (Jimenez et al., 1976). Of the crinanes identified and Newman, 2005; Pan et al., 2010). As such, over 60% of anti- in Boophone disticha, buphanisine 15 has been most studied, cancer agents presently in clinical use are derived from natural having been screened against 22 different cancers with the best sources, including plants, marine organisms and micro-organisms activity seen in Vinblastine-resistant oral epidermoid KB-V1 carci-

(Cragg and Newman, 2005; Pan et al., 2010). Examples of such noma cells [ED50 19.8 μg/mL (69.5 μM)] (Likhitwitayawuid et al., commercial drugs originating in plants include vinblastine, vin- 1993; Nair et al., 2012a). 6-Hydroxycrinamine 19, the only α- cristine, camptothecin, topotecan, irinotecan, etoposide and pacli- crinane of the compounds isolated, exhibited strong activity taxel (Cragg and Newman, 2005; Pan et al., 2010). In the South against mouse embryonic ﬁbroblasts (3T3) (MTD 0.63 μM) and African context, the work of Fouche et al. (2006, 2008) is most moderate activity against human neuroblastoma (SH-SY5Y) cells J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26 23

(IC50 54.5 μM) (Adewusi et al., 2012; Nair et al., 2012a). Activities Interestingly, forensic examination of an arrow taken from a for buphanidrine 13, buphanamine 11, crinamidine 17, crinine 3 San community in the Cape (Section 5) and known to have been and undulatine 14 were generally moderate to mild with crinine treated with Boophone disticha showed that the arrow poison was shown to be most effective against the multi-drug resistant human capable of killing mice within 20–30 min in subcutaneous doses of myeloid leukaemia cell line HL-60/Dox (IC50 14.04 μM) (Berkov 100–300 μg(De Smet, 1998; Mebs et al., 1996). Although animals et al., 2011; Nair et al., 2012a). Finally, out of a mini-panel of five and birds are known to consume various parts of the plant cancer cells, distichamine 16 exhibited best activity against HeLa (Table 1, entry 16), such tendencies have not been observed in cervical adenoacarcinoma (IC50 2.2 μM) (Nair et al., 2012a,c). humans (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). Information pertaining to in vivo studies of crinane alkaloids is However, the widespread usage of bulb concoctions, decoctions generally lacking in the literature; however, one study described and infusions suggest that these are obviously not taken at lethal significant reductions in the growth of sarcoma 180 ascites dosages (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). tumours in mice after treatment with various β-crinanes (Ghosal, In cases where poisoning with Boophone disticha were reported, 1986; Nair et al., 2012a). In relation to the mechanistic basis to the toxic effects produced included nausea, coma, muscular these alkaloids, the apoptotic mode of death in cancers has been flaccidity, visual impairment, stertorous breathing, respiratory demonstrated for crinine 3, distichamine 16, crinamine 26 and paralysis, feeble or increased pulse, dyspnoea, and hyperaemia haemanthamine 23, the first two of which are significant as they and oedema of the lungs (Watt and Breyer-Brandwijk, 1962; are constituents of Boophone disticha (Griffin et al., 2007; McNulty Nyazema, 1986; Hutchings et al., 1996; Du Plooy et al., 2001). et al., 2007; Van Goietsenoven et al., 2010; Berkov et al., 2011; Nair Acute oral toxicity studies revealed that extracts of Boophone et al., 2012c). For example, McNulty et al. (2007) showed that up to disticha were lethal to Sprague Dawley rats at doses equal to or 90% of rat hepatoma (5123 tc) cells exhibited apoptotic morphol- higher than 240 mg/kg, killing animals between 30 min and 3 h ogy after a 48 h treatment with either crinamine or haemanth- after dose administration, with an LD50 determined between 120 maine (ED50s 12.5 and 15 μM, respectively). Interestingly, this and 240 mg/kg (Gadaga et al., 2010). Furthermore, in the same activity was seen to be selective as normal human embryonic study the most prominent neurotoxicological effects linked to kidney (293t) cells remained unaffected by the treatment (Griffin doses higher than 240 mg/kg were increased flaccid limb paralysis, et al., 2007; McNulty et al., 2007; Nair et al., 2012a). Apart from retropulsion and hypoactivity which were evident from significant this Berkov et al. (2011) demonstrated via oligonucleosomal DNA rearing, reduced mobility and gait scores (Gadaga et al., 2010). In fragmentation that the cytotoxicity of crinine 3 in HL-60/Dox cells addition, it was suggested that mydriasis, palpebral closure,

(IC50 14.04 μM) ensues via the apoptosis pathway. In addition, tachypnoea, piloerection and spastic hind limb paralysis observed caspase-mediated apoptosis induction has recently been described at lower doses of Boophone disticha could be due to the antic- for distichamine 16 in lymphoblastic leukaemia (CEM) cells (IC50 holinergic effects of its alkaloid constituent lycorine 2 (Scheme 1) 4.5 μM) (Nair et al., 2012a,c). As such, ﬂow cytometric analysis (De Smet, 1996; Gadaga et al., 2010). The sub-acute toxic effects of showed that treatment with distichamine 16 increased the pro- Boophone disticha appear to be mediated via interference with the portion of G2/M phase cells in a dose-dependent manner, with neuronal pathways especially the central dopaminergic and motor concomitant reductions in the proportion of G0/G1 and S cells neurons (Gadaga et al., 2010). Histopathological observations (Nair et al., 2012a,c). In addition, the proportion of cells with sub- suggested the liver, small and large intestines, stomach, central

G1 amounts of DNA (apoptotic cells) increased (up to 23.7%) nervous system and peripheral nervous system as the target following a 24 h treatment with distichamine indicating that the organs (Gadaga et al., 2010). A further investigation was carried compound was capable of cell cycle disturbance and apoptosis out to ascertain whether pre-treatment of Boophone disticha induction (Nair et al., 2012a,c). Furthermore, distichamine induced poisoned BALB/c mice with a CNS acting serotonin antagonist a 12.5-fold increase in caspase-3/7 activity after 24 h at the highest (cyproheptadine) had a protective effect from toxicity and mor- tested concentration (20 μM) compared to untreated controls (Nair tality (Mutseura et al., 2013). Using the Functional Observational et al., 2012a,c). In addition to this, several changes in the expres- Battery (FOB) test to evaluate neurobehavioral and physiological sion of apoptosis-related proteins were detected via Western changes associated with toxicity, Mutseura et al. (2013) showed blotting in CEM cells following treatment with distichamine that cyproheptadine pre-treatment (15–20 mg/kg) led to a dose- (Nair et al., 2012a,c). For example, after 24 h at 10 and 20 mM dependent decrease in mortality from 80% in untreated animals to cleavage of PARP (89 kDa fragment) was observed which also 30% in the pre-treated group, as well as a reduction in other toxic corresponded with decreased levels of procaspase-3 (Nair et al., symptoms typically associated with Boophone disticha poisoning 2012a,c). Alongside this, expression of the tumour suppressor (Gadaga et al., 2010). protein p53 was discernible in the CEM cell control, and disticha- Apart from this, based on the putative effects of Boophone mine caused its enhanced expression after 24 h, notably at 10 and disticha on inflammatory response and immune function, extracts 20 mM(Nair et al., 2012a,c). No change in the expression of the of the plant were also investigated for ATP production effects in antiapoptotic protein Bcl-2 was observed but at 20 mM a decreased isolated human neutrophils (Botha et al., 2005). To this extent, level of the anti-apoptotic protein Mcl-1 was detected indicating ethanolic bulb extracts were shown to significantly decrease ATP the onset of apoptosis (Nair et al., 2012a,c). The traditional usage of as well as superoxide production in the neutrophil medium (Botha Boophone disticha in cancer therapy can therefore be ratified et al., 2005). A decrease in intracellular ATP is known to reflect through several sources of convincing pharmacological evidence. possible cell injury due to toxicity to neutrophils (Crouch et al., 1993). Conversely, it was suggested that the therapeutic benefits of 7.5. Toxicological studies Boophone disticha may reside with its ability to inhibit superoxide release from neutrophils (Botha et al., 2005). Studies on the Despite the manifold benefits associated with the usage of genotoxic effects of Boophone disticha showed extracts of the plant Boophone disticha in TM, the plant is nevertheless one of several to be genotoxic to human lymphocytic cells as determined by the South African Amaryllids classified as poisonous (Van Wyk et al., micronucleus test (Taylor et al., 2003). For example, in the test 2002, 2005). This is understandable given the long cultural history against aqueous methanol bulb extracts the number of micro- of Boophone disticha as an arrow poison (Watt and Breyer- nuclei produced per thousand binucleated cells in the lympho- Brandwijk, 1962; Bisset, 1989; De Smet, 1998; Hutchings et al., cytes was 8, 14 and 2 at set concentrations of 100, 500 and 1996). 2500 ppm, respectively, with a control value of 3 (Taylor et al., 24 J.J. Nair, J. Van Staden / Journal of Ethnopharmacology 151 (2014) 12–26

2003). Furthermore, no extracts of Boophone disticha exhibited these entities have been shown to be chieﬂy responsible for the anti-mutagenic effects as determined by the Ames test various biological effects exhibited by the plant, of which its use in (Verschaeve and Van Staden, 2008). Of the alkaloids identiﬁed in the traditional treatment of cancer is most striking. Despite this, Boophone disticha, lycorine 2 is known to exhibit toxic effects several areas of its usage in TM still require pharmacological (Watt and Breyer-Brandwijk, 1962; Van Wyk et al., 2002, 2005). validation. Furthermore, stringent efforts are required to further

For example, in dogs it exhibited an LD50 of 41 mg/kg and the delineate the well-documented toxicological properties of Boo- symptoms of poisoning included salivation, vomiting, diarrhoea phone disticha. As such, these collective efforts should go some and paralysis (Watt and Breyer-Brandwijk, 1962; Van Wyk et al., way towards maximising the potential of Boophone disticha as a 2002, 2005). In addition, subcutaneously administered lycorine beacon for the plant family Amaryllidaceae in South African induced nausea and emesis in beagle dogs starting at 0.5 mg/kg traditional medicine. and reaching statistical significance at 1 mg/kg (Kretzing et al., 2011). These symptoms were short lasting and occurred no later than 2.5 h post-treatment (Kretzing et al., 2011). In the same study Acknowledgements lycorine exhibited linear plasma kinetics with a mean elimination half-life of 0.67 h after a single subcutaneous dose and 0.3 h after Financial assistance in support of this work by the University of intravenous administration (Kretzing et al., 2011). Although lycor- KwaZulu-Natal is gratefully acknowledged. ine 2 is well-known within the Amaryllidaceae for its potent cytotoxic properties (Evidente and Kornienko, 2009; Lamoral- References Theys et al., 2010), it has nonetheless shown to be non-selective fi at this activity. For example, in a screen involving the ve human Adewusi, E.A., Fouche, G., Steenkamp, V., 2012. Cytotoxicity and acetylcholinester- cancer cells: acute lymphoblastic leukaemia (CEM), chronic mye- ase inhibitory activity of an isolated crinine alkaloid from Boophone disticha logenous leukaemia (K562), breast adenocarcinoma (MCF7), (Amaryllidaceae). J. Ethnopharmacol. 143, 572–578. Balunas, M.J., Kinghorn, A.D., 2005. 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