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Pharmacological potential and conservation prospect of (Hyacinthaceae) endemic to southern .

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Review Pharmacological potential and conservation prospect of the genus Eucomis (Hyacinthaceae) endemic to southern Africa

Nqobile A. Masondo, Jeffrey F. Finnie, Johannes Van Staden n

Research Centre for 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: The genus Eucomis (Hyacinthaceae) consists of 10 that are Received 22 August 2013 extensively used in African traditional medicine. This review is an appraisal of current information on the Received in revised form distribution and morphology, traditional uses, pharmacology, toxicology and approaches devised to 15 October 2013 enhance the conservation of the genus. Accepted 4 November 2013 Methods: A systematic and comprehensive literature search using electronic searches such as Scopus, Available online 15 November 2013 Google Scholar, Web of Science and ethnobotanical books was conducted. Keywords: Results: Evidence from traditional medicine usage shows wide utilization of this genus for ailments such Anti-inflammatory as respiratory, venereal diseases, rheumatism as well as kidney and bladder infections. Pharmacological Antimicrobial screening reported antimicrobial, antiplasmodial, antitumor, cytotoxic, phytotoxic and anti-inflammatory Medicinal plant properties. lily Conclusions: The potential of the genus Eucomis especially in terms of pharmacology cannot be over- Propagation fl Toxicology emphasized. Apart from the anti-in ammatory properties, the antifungal activity of Eucomis remains a valuable reservoir with potential application in the agriculture sector as a source of an affordable biocontrol agent. Based on the speculated toxic constituents in the genus Eucomis, it will be valuable to conduct detailed toxicological studies. Extensive utilization of members of the genus Eucomis is causing severe strain on wild populations.Althoughconventionalpropagationhasbeenrelativelyeffectiveinthealleviationofthedeclining status, micropropagation of members may be vital to guarantee the conservation of wild populations. & 2013 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction ...... 45 2. Distribution and general morphology of Eucomis species ...... 45 2.1. Distribution ...... 45 2.2. General morphology ...... 45 3. Documented uses in traditional medicine ...... 47 4. Phytochemistry and pharmacology of Eucomis species...... 49 4.1. Phytochemistry ...... 49 4.2. Pharmacology ...... 49 4.2.1. Anti-inflammatory screening...... 49 4.2.2. Antimicrobial screening...... 50 4.2.3. Other pharmacological properties...... 50 5. Safety and toxicity of Eucomis...... 50 6. Conservation status ...... 51 6.1. Conventional propagation ...... 52

Abbreviations: 2,4-D, 2,4-Dichlorophenoxy acetic acid; 2-iP, N6-Isopentenyladenine; ABA, abscisic acid; BA, benzyladenine; COX, cyclooxygenase; DCM, dichloromethane;

GA3, gibberellic acid; GA4þ 7,GA4 and GA7 gibberellin mixture; IAA, indole acetic acid; IBA, indole butyric acid; IUCN, Conservation of Nature and Natural Resource; MeJa, methyl jasmonate; MeOH, methanol; MIC, minimum inhibitory concentration; MS, Murashige and Skoog (1962); mT, meta-Topolin; NAA, α-naphthalene acetic acid; NSAIDs, non-steroidal anti-inflammatory drugs; PAA, phenylacetic acid; PBZ, paclobutrazol; TPA, 12-O-tetradecanoylphorbol 13-acetate n Corresponding author. Tel.: þ27 33 2605130. E-mail address: [email protected] (J. Van Staden).

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.11.002 N.A. Masondo et al. / Journal of Ethnopharmacology 151 (2014) 44–53 45

6.2. Micropropagation of Eucomis ...... 52 7. Conclusions ...... 52 Acknowledgments...... 52 References...... 52

1. Introduction development depend on certain factors such as climatic regions and environmental conditions. For instance, is The genus Eucomis is a member of the Hyacinthaceae (formerly commonly found at the base of the cliffs along moist known as Liliaceae) comprising of 41 genera distributed in Europe, slopes ranging from 1800 to 2600 m while Eucomis humilis is South America and Africa. In southern Africa, there are 27 genera found on slopes that range from 2400 to 2900 m. Eucomis and approximately 200 species found in the Cape Floristic Region, autumnalis is distributed in damp craters in grasslands that range South Africa (Pooley, 2005). Among the genera in southern Africa from 2100 to 2400 m (Trauseld, 1969). The specificity of Eucomis Eucomis is endemic to the region. The genus is relatively small, species to certain environmental and climatic conditions are consisting of 10 species (Pooley, 2005). Although Eucomis species amongst the contributing factors that have resulted in the exces- are generally summer blooming, is winter blooming. sive decline and vulnerable status of the species in South Africa The species comprise of deciduous geophytes with long and (Raimondo et al., 2009). narrow leaves topped with densely packed flowers (Compton, 1990). The characteristic leaf-like at the peak of the flower 2.2. General morphology spikes earned this genus its Greek name ‘eukomes’ which refers to ‘beautiful headed’ (Bryan, 1989). Eucomis is commonly called Eucomis species are geophytes with ovoid or globose shaped ‘pineapple lily’ because of the flower spikes that resemble pine- comprising of hard cortices (Bryan, 1989; De Hertogh and Le apples (Pienaar, 1984). Nard, 1993). The size ranges from 50 to 150 mm in diameter In traditional medicine, Eucomis species are widely utilized and have a perceptible horizontal striped base with brown to black against various ailments including respiratory, venereal diseases scales (Fig. 1B). The inner part of the bulb is yellow-white and and rheumatism (Hutchings et al., 1996). Consequently, Eucomis turns black when exposed to air (Mander et al., 1995). The bulbs species have been evaluated in both in vitro and in vivo bioassays have branched perennial fleshy contractile roots with root hairs for anti-inflammatory, antibacterial, antihistaminic and angio- (Fig. 1A). They are characterized by a rosette of smooth often shiny protective potentials (Heller and Tamm, 1981; Amschler et al., leaves that are lanceolate, elliptic or ovate and bend backwards 1996). The extensive biological activities of Eucomis species have (Fig. 1C). been mainly attributed to phytochemicals such as the homoiso- The stem ranges from 30 to 100 cm in height depending on the flavanones commonly present in the plant. An in-depth review on species. A straight cylindrical inflorescence with a pale lime-green the phytochemistry of the genus Eucomis is documented flower is located at the top of the flowering stem (Koorbanally et al., 2006a). The extensive exploitation of Eucomis (De Hertogh and Le Nard, 1993). The flower colour varies from species in traditional medicine together with its slow propagation yellowish-green or white with margins varying from pale to dark rate has inevitably resulted in the decline of the majority of purple. Flower color turns green on maturity (Fig. 1D). After species, of which some are endangered/threatened (Raimondo and fertilization, green or brown fruits appear contain- et al., 2009). The current review focuses on the pharmacological ing dull blackish-brown seeds (Bryan, 1989; Du Plessis and characteristics of the genus Eucomis. A critical evaluation of avai- Duncan, 1989). lable propagation protocols as a possible means of enhancing their The most common feature used for plant identification is conservation status is discussed. Even though members of the flower morphology. However, the aforementioned method is genus Eucomis are widely utilized globally, the current review is difficult with the genus Eucomis because the flowers are morpho- presented from a South African perspective. logically similar. Therefore, features such as fragrance, plant size and leaf color allows for better differentiation among species. Members of the genus emit distinct floral scents. Sweet aroma 2. Distribution and general morphology of Eucomis species species include Eucomis amaryllidifolia, , , Eucomis grimshawii, Eucomis pallidiflora and 2.1. Distribution while Eucomis bicolor, Eucomis humilis, , Eucomis regia, Eucomis schijffii and Eucomis Eucomis genus is widely exploited for its medicinal and horti- vandermerwei emit an unpleasant scent (Zonneveld and Duncan, cultural value in southern African countries such as South Africa, 2010). The presence or absence of a purple color at the leaf base or , , Swaziland, and (Pienaar, flower and the cylindrical shape of the scape are used to distin- 1984; Du Plessis and Duncan, 1989). In South Africa, Eucomis guish Eucomis species. Taken together, these characteristics species are distributed across all the nine Provinces. In terms of become more difficult for identification and differentiation among species richness, Eastern Cape with nine species is best repre- closely-related species or subspecies. Therefore the use of genome sented, followed by KwaZulu-Natal with seven species while size together with nuclear DNA content provides a better tool to Northern Cape is the least endowed Province with only one distinguish species. species (Table 1). The distribution pattern of the genus Eucomis According to Reyneke and Liebenberg (1980), Eucomis species across the Provinces varies depending on the species. While some have 15 chromosomes which exist either in diploid (small species) or species occur in two or more Provinces, other species such as tetraploid (larger species) states. The diploid species (2n¼2x¼30) are Eucomis humilis, Eucomis montana, Eucomis pallidiflora subspecies Eucomis amaryllidifolia, Eucomis bicolor, Eucomis grimshawii, Eucomis pole-evansii and Eucomis zambesiaca are found only in one Pro- regia, Eucomis schijffii, and Eucomis zambesiaca vince. This is a demonstration of their specificity to particular while the tetraploid species (2n¼4x¼60) include Eucomis autum- climatic conditions within South Africa (Table 1). Their growth and nalis, Eucomis comosa, Eucomis humilis, Eucomis montana and Eucomis 46 N.A. Masondo et al. / Journal of Ethnopharmacology 151 (2014) 44–53

Table 1 Distribution and ethnobotanical uses of members of the genus Eucomis found in South Africa.

Species Province Plant Traditional uses References (s) part(s)

Eucomis autumnalis (Mill.) Chitt. FS, KZN, Leaves, Colic, flatulence, kidney and bladder problems, Bisi-Johnson et al. (2010), (synonym Eucomis undulate) EC, M, G bulbs, nausea, coughs syphilis, abdominal distension Hutchings et al. (1996), Roberts (1990), roots Watt and Breyer-Brandwijk (1962) Eucomis autumnalis (Mill.) Chitt. L, FS, EC NA NA NA subspecies amaryllidifolia (Baker) Reyneke Eucomis autumnalis (Mill.) Chitt. EC, L Leaves, Administered as enemas to treat lower backache, biliousness, Hutchings et al. (1996) subspecies autumnalis bulbs urinary diseases, post-operative recovery, fevers and fractures Eucomis autumnalis (Mill.) Chitt. KZN, FS, Bulbs Administered as enemas to treat lower backache, biliousness, Hutchings et al. (1996) subspecies clavata (Baker) Reyneke M, G, L, urinary diseases, post-operative recovery, fevers and fractures NW Eucomis bicolor Baker EC, KZN Bulbs Colic and purgative Watt and Breyer-Brandwijk (1962), Hutchings et al. (1996) Eucomis comosa (Houtt.)Wehrh. EC, KZN Bulbs, For rheumatism, teething infants and purgative Cunningham (1988), Hutchings et al. (synonym Eucomis punctate) roots (1996), Watt and Breyer-Brandwijk (1962) Eucomis comosa (Houtt.)Wehrh. WC, EC, Bulbs, Rheumatism and teething in infants Watt and Breyer-Brandwijk (1962) variety comosa KZN roots Eucomis comosa (Houtt.)Wehrh. EC, L NA NA NA variety striata (Don) Willd. Eucomis humilis Baker KZN NA NA NA Eucomis montana Compton M NA NA NA Eucomis pallidiflora Baker subspecies EC, M Bulbs Mental diseases Watt and Breyer-Brandwijk (1962) pallidiflora Eucomis pallidiflora subspecies M Bulbs Erectile dysfunction, tuberculosis, blood clotting, Semenya and Potgieter (2013), Semenya pole-evansii (N.E.Br.) cough et al. (2013) Reyneke ex J. C. Manning Eucomis regia L’Hér. NC, WC Bulbs, Venereal diseases, diarrhea, cough, biliousness Watt and Breyer-Brandwijk (1962) roots and prevent premature childbirth Eucomis schijffii Reyneke EC, KZN Bulbs Venereal diseases, diarrhea, coughs and used Watt and Breyer-Brandwijk (1962) as enema for biliousness, prevention of pre-mature birth, lower back pains Eucomis vandermerwei I. Verd. M, L NA NA NA Eucomis zambesiaca Baker L NA NA NA

NA—Not Available, EC—Eastern Cape, FS—Free State, G—Gauteng, KZN—KwaZulu-Natal, L—Limpopo, M—Mpumalanga, NC—Northern Cape, NW—North West, WC—Western Cape.

Fig. 1. Eucomis morphology A-roots; B-bulb; C-whole plants; D-inflorescence. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.) N.A. Masondo et al. / Journal of Ethnopharmacology 151 (2014) 44–53 47

Table 2 Example of in vitro studies screening different Eucomis species for anti-inflammatory activity.

Species Extracting Indomethacin Finding(s) and extract concentration Reference(s) solvent inhibition (concentration)

Eucomis autumnalis Ethanol, 66. 5% inhibition High COX enzyme inhibition in bulb extracts from ethanol (90%) at 0.5 mg/ml and Jäger et al. (1996) water (0.5 mg) water (73%) at 0.5 mg/ml Eucomis autumnalis NR NR Bulb extract showed 88% inhibition Gaidamashvili and Van Staden (2006) Eucomis autumnalis 70% 64.18% inhibition Acetone extracts at 250 mg/ml had Z75% enzyme inhibition except for smoke-water Ndhlala et al. acetone, (5 mM) (COX-1), the control and light exposure treatment (COX-2) (2012) water Eucomis autumnalis Ethanol, Inhibition NR (5 mM) COX-1 activity from ethanol at 250 mg/ml and water extract at 500 mg/ml was Taylor and Van subspecies water moderate (40–70%) Staden (2001b) amaryllidifolia Eucomis autumnalis Ethanol Inhibition Ethanol bulb extract at 250 mg/ml exhibited high activity against COX-1 and COX-2 Taylor and Van subspecies NR (COX-1¼5 mM) inhibitors (70–100%) Staden (2002b) amaryllidifolia NR (COX- 2¼200 mM)

Eucomis autumnalis Ethanol, Inhibition NR (5 mM) IC50 values COX-1 from ethanol extracts at 250 mg/ml were evaluated. IC50 value for Taylor and Van subspecies water leaf extract was 15, for bulb extract was 72 and for root extract was 27 g/ml Staden (2001b) autumnalis Eucomis autumnalis Ethanol Inhibition Ethanol bulb extracts at 250 mg/ml exhibited high COX-1 and COX-2 activity Taylor and Van

subspecies NR (COX-1¼5 mM) (70–100%). IC50 ratio for COX-1 and COX-2 was 1.9 (leaf), 0.8 (bulb) and 0.7 (root) Staden (2002b) autumnalis NR (COX- 2¼200 mM) Eucomis autumnalis Ethyl 80% inhibition Bulb and root extracts at 250 mg/ml had higher inhibitory activity (490%) than leaf Zschocke et al. subspecies acetate, (20 mM) extract (65%) against COX-1 (2000) autumnalis hexane Eucomis autumnalis Ethanol, 64% inhibition Both ethanol extracts at 50 mg/ml of fresh and stored plant materials exhibited 100% Stafford et al. subspecies water (50 mg/ml) COX-1 inhibition while water extracts showed r37% inhibition (2005) autumnalis Eucomis autumnalis Ethanol, Inhibition NR (5 mM) COX-1 activity from ethanol at 250 mg/ml and water extract at 500 mg/ml was Taylor and Van subspecies clavata water moderate (40–70%) Staden (2001b) Eucomis autumnalis Ethanol Inhibition Ethanol bulb extract at 250 mg/ml exhibited high activity against COX-1 and COX-2 Taylor and Van subspecies clavata NR (COX-1¼5 mM) inhibitors (70–100%) Staden (2002b) NR (COX- 2¼200 mM) Eucomis bicolor Ethanol, Activity NR (5 mM) COX-1 activity from leaf ethanol at 250 mg/ml and water extracts at 500 mg/ml, ethanol Taylor and Van water bulb extract was high (70–100) Staden (2001b) Eucomis comosa Ethanol Inhibition Ethanol bulb extract at 250 mg/ml exhibited high activity against COX-1 and COX-2 Taylor and Van NR (COX-1¼5 mM) inhibitors (70–100%) Staden (2002b) NR (COX- 2¼200 mM) Eucomis comosa Ethanol, Inhibition NR (5 mM) COX-1 activity from ethanol at 250 mg/ml and water extract at 500 mg/ml was Taylor and Van subspecies comosa water moderate (40–70%) Staden (2001b) Eucomis comosa Ethanol Inhibition Ethanol bulb and root extract at 250 mg/ml exhibited high activity against COX-1 Taylor and Van subspecies comosa NR (COX-1¼5 mM) inhibitors (70–100%) Staden (2002b) NR (COX- 2¼200 mM) Eucomis comosa Ethanol, Inhibition NR (5 mM) COX-1 activity from ethanol 250 mg/ml and water extract 500 mg/ml was moderate Taylor and Van subspecies striata water (40–70%) Staden (2001b) Eucomis comosa Ethanol Inhibition Ethanol bulb extract 250 mg/ml exhibited high activity against COX-1 and COX-2 Taylor and Van subspecies striata NR (COX-1¼5 mM) inhibitors (70–100%) Staden (2002b) NR (COX- 2¼200 mM) Eucomis humilis Ethanol, Inhibition COX-1 and COX-2 activity from ethanol 250 mg/ml and water bulb extracts 500 mg/ml Taylor and Van water NR (COX-1¼5 mM) was high (70–100%) Staden (2002b) NR (COX- 2¼200 mM) Eucomis zambesiaca Ethanol, Inhibition NR (5 mM) COX-1 activity from ethanol at 250 mg/ml and water extract 500 mg/ml was moderate Taylor and Van water (40–70%) Staden (2001b) Eucomis zambesiaca Ethanol Inhibition Ethanol bulb and root extract at 250 mg/ml exhibited high COX-1 activity (70–100%) Taylor and Van NR (COX-1¼5 mM) Staden (2002b) NR (COX-2¼ 200 mM)

NR—Not reported.

pallidiflora (Zonneveld and Duncan, 2010). However, based on the 3. Documented uses in traditional medicine variation in chromosome number of individual tetraploid species, Reyneke and Liebenberg (1980) concluded that tetraploids are in fact As highlighted in Table 1, several Eucomis species are utilized allotetraploids. A detailed review focusing on the genome size of the as a remedy against various ailments in traditional medicine. different Eucomis species is available (Zonneveld and Duncan, 2010). In South Africa, plant materials are often prepared as decoctions, 48 Table 3 Example of studies screening different South African Eucomis species for antimicrobial activity.

Species Extracting solvent Test system and organism(s) Positive control activity Finding(s) References

Eucomis autumnalis Methanol, water In vitro – five bacterial strains 10 ml of neomycin (200–500 mg/ml) was used in Methanol bulb extract at 1 mg/ml extract exhibited a ratio Rabe and Van each petri-dish in the agar diffusion assay of 0.13 zone of inhibition against Bacillus subtilis when Staden (1997) compared to the positive control Eucomis autumnalis Ethanol, ethyl In vitro – two clinical and one Amphotericin B MIC activity 1.56 mg/ml Bulb extracts had minimum inhibitory Motsei et al. acetate, hexane, standard Candida albicans concentration (MIC) 8.35 mg/ml (2003) water Eucomis autumnalis Methanol, ethyl In vitro – two Escherichia coli Kanamycin with an MIC of 0.195 mg/ml Ethyl acetate extract had an MIC of 0.27 mg/ml Bisi-Johnson acetate, butanol, strains et al. (2011) water Acetone bulb extracts had an MIC of 0. 78 mg/ml Ndhlala et al. Eucomis autumnalis 70% acetone, water In vitro – four bacterial strains Neomycin MIC activity B. subtilis 3 Staphylococcus aureus 6.125 10 3 against B. subtilis and S. aureus (2012) and Candida albicans 1.531 10 Eucomis autumnalis Acetone, methanol, In vitro – 94 microbial strains 27 different antibiotic were used for the assay The leaf extracts were not active at the highest tested Mohlakoana water concentration (20 mg/ml) (2010) 44 (2014) 151 Ethnopharmacology of Journal / al. et Masondo N.A. @ Eucomis autumnalis Methanol In vitro – seven plant fungal Carbendazim/difenoconazole (Eria – 187.5 g/l EC) About Whole plant extract at 100 mg/ml had high inhibition Eksteen et al. subspecies strains 100% inhibition against three of the strains (1 mg/ml) against Brotryosphaeria dothidea (85%) and Pythium ultimum (2001) clavata fungicide (95.4%) @ Eucomis autumnalis Methanol In vitro – eight plant fungal Carbendazim/difenoconazole (Eria – 187.5 g/l EC) About Bulb extracts at 1 mg/ml had a significant (Z73%) growth Pretorius et al. subspecies strains 100% inhibition against two of the strains (1 mg/ml) inhibition against six of tested fungal strains (2002) clavata @ Eucomis autumnalis Methanol In vivo – one plant fungal strain Carbendazim/difenoconazole (Eria – 187.5 g/l EC) 1 mg/ml Extract concentration of 1 mg/ml prevented spore infection Pretorius et al. subspecies prevented spore infection (1 mg/ml) (Mycosphaerella pinodes) in pea plant (2002) clavata Eucomis comosa Methanol In vitro – one bacterial strain Neomycin MIC activity 0.0025 mM Compounds 1 and 2 had high inhibitory activity against S. aureus Du Toit et al. with MIC value r0.52 mM. Compound 9 (0.98 mM) had (2007) better inhibitory activity compared to compound 8 (4.15 mM). Compound 8 showed bacteriostatic activity (2.07 mM)

NR—Not reported, Compounds (1)¼1, 7-hydroxy-5-methoxy-3-(4′-hydroxybenzyl)-4-chromanone; Compounds (2)¼5,7-dihydroxy-8-methoxy-3 (4′ hydroxybenzyl)-4-chromanone (3,9-dihydropunctatin) Compounds (8)¼scillascillin; (9)¼23S-17α,23-epoxy-3β,28,29-trihydroxy-27-norlanost-8-en-24-one. – 53 N.A. Masondo et al. / Journal of Ethnopharmacology 151 (2014) 44–53 49 infusions and enemas. Moreover, the Zulu, Tswana, Sotho and found in the Hyacinthoideae genera have been isolated from six Xhosa people commonly use either water or milk for the preparing Eucomis species (Pohl et al., 2000). However, eight species including of these decoctions. Eucomis autumnalis subspecies amaryllidiflolia, Eucomis autumnalis Evidence shows that bulbs of Eucomis species are the most subspecies clavata, Eucomis comosa subspecies striata, Eucomis pallidi- utilized plant part when compared to other organs such as roots, flora subspecies pallidiflora, Eucomis regia, Eucomis humilis, Eucomis stems and leaves (Table 1). However, bulbs and roots are occa- vandermerwei and Eucomis zambesiaca require more studies for sionally combined as ingredients in infusions for alleviation of possible isolation of novel compound(s). Recently, the phytochemical pain and fever (Hutchings et al., 1996). It has also been documen- content of members of Eucomis has been extensively reviewed ted that the Zulu tribe use bulb infusions for the relief of (Koorbanally et al., 2006a, 2006b). biliousness, enhancing sexual prowess and cleansing of blood The presence and wide diversity of flavonoids in Eucomis (Mander et al., 1995). Based on documented uses as summarized species have been associated with their pharmacological proper- in Table 1, it is apparent that the majority of the Eucomis species ties, for example, anti-inflammatory activity (Heller and Tamm, are highly valued in traditional medicine as demonstrated in their 1981). Often pain and inflammation are common underlying numerous uses e.g. treating kidney and bladder ailments as well as symptoms in the majority of ailments treated with Eucomis species nausea and coughs. In folk medicine, it is believed that bulbs (Koorbanally et al., 2005). The large number of isolated com- possess mysterious power and they are used as protective charms pounds from Eucomis plants is an indication of the value of the (Watt and Breyer-Brandwijk, 1962). genus as a potential candidate for new drugs in the pharmaceu- Different plant species are combined and used together as a tical industry for pain related ailments and bacterial/fungal infec- common practice in traditional medicine. Along this line, Eucomis tions (Table 3). with Crinum, Bowiea, Xanthoxylum and Becium are combined and used as a form of treatment against cancer (Fennell and Van 4.2. Pharmacology Staden, 2001). The leaves are used as a poultice for sores and boils and are wrapped around the wrists to reduce fever. Apart from the In an attempt to rationalize the wide usage and validate the excessive use of the plant in the treatment of human diseases, efficacy of medicinal plants, researchers have been evaluating Eucomis species also serve as a remedy for animal ailments. The bioactivity under laboratory conditions. Eucomis species have been leaves and bulbs of Eucomis are combined with Medicago sativa or mainly screened for their anti-inflammatory and antimicrobial Zea mays leaves to treat gall sickness and other diseases in cattle activities. Based on the numerous benefits associated with in vitro (Roberts, 1990). Moreover, the plant is used for the treatment of test systems, it remains popular and a widely-used approach by venereal diseases in livestock (Watt and Breyer-Brandwijk, 1962; researchers (Houghton et al., 2007). The majority of studies Hutchings et al., 1996). documented in the current review were conducted using in vitro methods (Tables 2–4).

Eucomis 4. Phytochemistry and pharmacology of species 4.2.1. Anti-inflammatory screening Inflammation processes involve the production of prostaglandins The extensive traditional use of Eucomis species has led to which are highly active pro-inflammatory mediators (Zschocke et al., several pharmacological properties being evaluated (Table 2). The 2002). The biosynthesis of prostaglandin is regulated by cycloox- increasing number of ethnopharmacological studies has shown ygenase (COX) enzymes (Jäger and Van Staden, 2005). The enzyme the potential substitution and supplementation of synthetic drugs occurs in two major isoforms namely COX-1 and COX-2. While COX-1 with extracts and/or isolated compounds from medicinal plants contributes to the homeostasis of numerous physiological functions (Rates, 2001; Newman et al., 2003). Evidently, the phytochemical in different tissues, COX-2 is involved in several inflammatory diversity in higher plants accounts for their promising pharmaco- reactions caused by inflammatory stimuli such as mitogens and logical potential. cytokinesis (O’Banion et al., 1991; Kujubu et al., 1991). In an effort to alleviate inflammation, several plant extracts have been screened 4.1. Phytochemistry in vitro for prostaglandin synthesis inhibition. In traditional medicine, Eucomis species are commonly utilized for inflammation and pain Several classes of phytochemicals have been isolated from Eucomis related ailments (Table 1). Thus, the majority of the pharmacological species including homoisoflavanones, spiocyclic nortriterpene, benzo- screenings have focused on their anti-inflammatory potential pyranones amongst others. Approximately 39 constituents commonly (Table 2). Varying levels of anti-inflammatory activity have been

Table 4 Other in vitro activities of South African Eucomis species.

Species Extracting solution Bioactivity Positive control activity Report on the activity References (concentration)

Eucomis autumnalis Dichloromethane, Antiplasmodial Chloroquine diphosphate The bulb extracts from Dichloromethane (70 mg/ml), Clarkson Dichloromethane: activity (NR) Dichloromethane:Methanol (9.5 mg/ml) and water (100 mg/ml) et al. (2004)

Methanol (1:1) and had IC50 values against Plasmodium falciparum water

Eucomis autumnalis Methanol Cytotoxicity Berberine IC50 ¼9.8 mg/ml The methanol extracts were cytotoxic with an IC50 value of Bisi-Johnson activity 7.8 mg/ml et al. (2011) Eucomis autumnalis Methanol Phytotoxicity Carbendazim/difenoconazole Up to 2 mg/ml of bulb extract showed no phytotoxic effect Pretorius subspecies (Eria@ – 187.5 g/l EC) NR et al. (2002) clavata (1 mg/ml)

NR—Not Reported. 50 N.A. Masondo et al. / Journal of Ethnopharmacology 151 (2014) 44–53 detected in several Eucomis species. Even though the majority of dothidea, Pythium ultimum and Candida albicans have been investi- species exhibited high COX-1 or COX-2 enzyme inhibition, Eucomis gated (Table 3). Eucomis extracts inhibited only a few bacterial strains autumnalis, Eucomis autumnalis subspecies autumnalis, Eucomis such as Bacillus subtilis, Escherichia coli and Staphylococcus aureus. autumnalis subspecies amaryllidiflolia and Eucomis humilis were the In a recent study (Bisi-Johnson et al., 2011), ethyl acetate extracts of most active. Among researchers, there are concerted efforts at Eucomis autumnalis showed remarkable minimum inhibitory concen- discovering COX-2 preferential inhibitors as a result of the numerous tration (MIC) activity (0.27 mg/ml) against Escherichia coli.Further- side effects associated with COX-1 inhibitors (Wallace and Chin, more, Ndhlala et al. (2012) showed the activity of Eucomis autumnalis 1997). Therefore, the selective inhibition of COX-2 by Eucomis against Bacillus subtilis and Staphylococcus aureus with an MIC value autumnalis subspecies autumnalis extract is worth pursuing for of 0.78 mg/ml. Generally, compounds are often isolated from crude possible isolation of such desired bioactive compound(s). extracts with antimicrobial potential. Five compounds isolated from In addition to the well-known effects of species-type on the anti- Eucomis comosa and Eucomis schijffii showed significant MIC values inflammatory activity, stringent studies aimed at the better under- (0.52 and 0.24 mM) against Staphylococcus aureus (Du Toit et al. standing of other crucial factors affecting Eucomis species inhibition (2007). Some of the compounds isolated by the authors include of COX have been conducted. In relation to extracting solvent, many (1)¼1, 7-hydroxy-5-methoxy-3-(4′-hydroxybenzyl)-4-chromanone; of the evaluated Eucomis species exhibited high COX-1 and COX-2 (2)¼5,7-dihydroxy-8-methoxy-3 (4′ hydroxybenzyl)-4-chromanone activity when extracted with ethanol or 70% acetone as compared (3,9-dihydropunctatin); (8)¼scillascillin and (9)¼23S-17α,23-epoxy- to water extracts (Table 2). Generally water extracts are known 3β,28,29-trihydroxy-27-norlanost-8-en-24-one. to exhibit lower activity when compared to non-polar extracts in When Eucomis was tested against Candida albicans the extracts various pharmacological studies. However, Jäger et al. (1996) showed showed a MIC value of Z1.56 mg/ml (Motsei et al., 2003; Ndhlala that extracting solvents had no significant effect on the anti- et al., 2012). In other studies, Eucomis autumnalis extracts were inflammatory activity of Eucomis autumnalis. This was observed from not effective against Candida albicans strains (Mohlakoana, 2010). the high activity of prostaglandin-synthesis inhibition in water (73%) Although Eucomis species were not very effective against Candida and ethanol (90%) extracts of Eucomis autumnalis at 0.5 mg/ml albicans, the plants may be effective against other fungal strains. concentration. Furthermore, the majority of the anti-inflammatory Therefore further research needs to be conducted on the anti- studies on the Eucomis genus have shown the potency of bulb fungal properties of Eucomis species using other fungal strains, extracts against both COX-1 and COX-2 enzymes (Table 2). However, which if effective, can then be further tested in vivo. As a potential leaf extracts of species such as Eucomis bicolor (Taylor and Van biocontrol agent, Eucomis autumnalis subspecies clavata exhibited Staden, 2001b)andEucomis autumnalis subspecies autumnalis (Taylor significantly high antifungal activity against seven plant pathogens and Van Staden, 2002b) showed higher enzyme inhibitory activity (Eksteen et al., 2001). In addition, Eucomis autumnalis subspecies compared to the bulb extracts. In addition, COX-2/COX-1 ratio for clavata extracts had Z73% inhibition against six plant pathogens leaves, bulb and root showed that leaf extracts (1.9) were more in a field trial (Pretorius et al., 2002). The potential of Eucomis effective against COX-1 while bulbs and root were effective against autumnalis subspecies clavata against plant pathogens is note- COX-2enzyme(0.8and0.7respectively).ThehighCOX-1activity worthy as it could provide an affordable and accessible means of depicted by leaf extracts of Eucomis autumnalis subspecies autumnalis controlling plant pathogens in agriculture. create an awareness of the efficacy in other plant parts besides the vulnerable bulbs. The preferential COX-2 inhibition by bulb and root 4.2.3. Other pharmacological properties extracts of Eucomis autumnalis subspecies autumnalis is an indication Besides the aforementioned pharmacological properties, Eucomis of its pharmaceutical potential (Taylor and Van Staden, 2002b). has been tested for cytotoxicity, phytotoxicity, anticancer and anti- The increased demand for Eucomis plants in both formal and plasmodial activities (Table 4). Eucomis had noteworthy antitumor and informal markets has exacerbated harvesting from wild populations. cytotoxicity activity. Eucomis autumnalis was evaluated for cytotoxicity In order to prevent plant pathogens from attacking fresh plant and the methanol extracts showed good activity (IC50 7.8 mg/ml) materials, plants are dried, stored and sold later. Due to such against the human hepatoma cell line (Huh-7) compared to the practices, there is increasing concern on the pharmacological potency positivecontrolwithanIC50 of 9.8 mg/ml (Bisi-Johnson et al., 2011). of dried medicinal plants as compared to fresh plant materials. When Eucomis was screened for antitumor activity (Mimaki et al., Several authors have reported variation in anti-inflammatory activity 1994), eucosterol glycoside isolated from Eucomis bicolor showed 44% among stored and fresh plant material (Fennell et al., 2004). Stafford inhibition of 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated et al. (2005) showed the effectiveness of both stored (90 days) and 32P incorporation into phospholipids of HeLa against tumor-promo- fresh Eucomis autumnalis extracts with 100% inhibition. Furthermore, ters. In vivo tests are essential for further validation of Eucomis extracts Taylor and Van Staden (2002a) reported high COX-1 activity (approxi- as anti-cancer agents. This may be of great value in the search for mately 70%) from Eucomis autumnalis subspecies autumnalis stored anticancerdrugswithpotentiallessersideeffectsascomparedto (dormant) in cold conditions (10 1C) compared to those maintained at other synthetic drug treatments. Furthermore, when Eucomis autum- 15–24 1C(55%). nalis were evaluated for phytotoxicity the bulb extract (up to 2 mg/ml) were not toxic to pea leaves (Pretorius et al., 2002)and1mg/ml inhibited spore germination. 4.2.2. Antimicrobial screening Excessive increases in drug resistance and side effects with the frequently used medications (mainly antibiotics) are well-known. 5. Safety and toxicity of Eucomis Consequently, enormous efforts have been geared towards the screen- ing of medicinal plants as a potential source of novel leads in the The issues of quality control and safety of conventional drugs treatments of microbial infections (Rates, 2001; Taylor et al., 2001). are important aspects of pharmaceutical industries. In traditional Different plant parts and extracting solvents have been used in the medicine, however, there is limited information on the safety of in vitro and in vivo screening of several Eucomis species. Effect of plant extracts or herbal products because of the presumed safety Eucomis extracts on diverse microbes such as Staphylococcus aureus, of natural products. Even though the Eucomis genus is extensively Staphylococcus epidermis, Bacillus subtilis, Klebsiella pneumonia, Escher- utilized in traditional medicine it has been implicated in human ichia coli, Botrytis cinerea, Fusarium oxysporum, Mycosphaerella pinodes, poisoning and death in sheep (Watt and Breyer-Brandwijk, 1962). Sclerotium rolfsii, Rhizoctonia solani, Vericillium dahlia, Brotryosphaeria According to Hutchings et al. (1996) abdominal pain, diarrhea and N.A. Masondo et al. / Journal of Ethnopharmacology 151 (2014) 44–53 51

Table 5 Conservation status and propagation protocols of Eucomis species.

Species name Conservation Explants Media Results References status

Eucomis Declining Bulb twin-scale, MS media, BA, NAA, BA, BA and NAA showed significant shoot regeneration Taylor and Van Staden autumnalis bulb-scale, shoots, NAA:BA, IAA, IBA (2001a), Ault (1995) leaf Eucomis Not evaluated Bulb, root, leaf MS media, NAA:BA, Optimum shoot initiation was obtained from 1 mg/l: 1 mg/l Taylor and Van Staden autumnalis IAA, IBA (NAA:BA) with no shoots initiated from 1 mg/l:2 mg/l media. (2001a) subspecies Root initiation was achieved from 1 mg/l NAA amaryllidifolia Eucomis Not evaluated Bulb, root, leaf, 1/10th MS media, Seed germinated in dark conditions and when treated with Kulkarni et al. (2006), autumnalis seed Smoke-water and smoke-water or 3-methyl-2H-furo[2,3-c]pyran-2-one. Taylor and Van Staden subspecies smoke-compound, Optimum shoot initiation obtained from 1 mg/l:1 mg/l (2001a) autumnalis NAA:BA, IAA, IBA (NAA:BA). Root initiation was achieved from 1 mg/l NAA Eucomis Not evaluated Bulb, root, leaf MS media, NAA:BA, Optimum shoot initiation was obtained from 1 mg/l:1 (NAA: Taylor and Van Staden autumnalis IAA, IBA BA) with no shoots initiated from 1 mg/l:2 mg/l media. Root (2001a) subspecies initiation was achieved from 1 mg/l NAA clavata Eucomis bicolor Near Bulb, root, leaf MS media, NAA:BA, Optimum shoot initiation was obtained from 1 mg/l:2 mg/l Taylor and Van Staden threatened IAA, IBA (NAA:BA) Root initiation was achieved from 1 mg/l IBA (2001a) Eucomis comosa Declining Bulb twin-scale, MS media, BA, NAA Optimum shoot initiation was obtained from 1 mg/l:1 mg/l Ault (1995), Taylor and bulb-scale, root, (NAA:BA). Van Staden (2001a) leaf BA and NAA had significant shoot regeneration Eucomis comosa Not evaluated Bulb, root, leaf MS media, NAA:BA, Optimum shoot initiation was obtained from 1 mg/l:2 mg/l Taylor and Van Staden subspecies IAA, IBA (NAA:BA). Root initiation was achieved from 1 mg/l IBA (2001a) comosa Eucomis comosa Not evaluated Bulb, root, leaf MS media, NAA:BA, Optimum shoot initiation was obtained from 1 mg/l:1 mg/l Taylor and Van Staden subspecies IAA, IBA (NAA:BA). Root initiation was achieved from 1 mg/l NAA (2001a) striata Eucomis humilis Least concern Bulb, root, leaf MS media, NAA:BA, Optimum shoot initiation was obtained from 1 mg/l:1 mg/l Taylor and Van Staden IAA, IBA (NAA:BA). Root initiation was achieved from 1 mg/l IBA (2001a) Eucomis montana Declining NR NR NR NR Eucomis Least concern NR NR NR NR pallidiflora subspecies pallidiflora Eucomis Near NR NR NR NR pallidiflora threatened subspecies pole-evansii Eucomis regia Least concern NR NR NR NR Eucomis schijffii Least concern NR NR NR NR Eucomis Vulnerable Shoots, leaf MS media, BA, IAA Successful shoot initiation was obtain from explants McCartan et al. (1999) vandermerwei propagated at 1 to 2 mg/l of BA and 1 mg/l IAA Eucomis Least concern Bulb twin-scale, MS media, BA, NAA, Significant shoot regeneration was obtained from NAA at Ault (1995), Cheesman zambesiaca bulb-scale, root, NAA:BA, IAA, IBA, 5.4 mM et al. (2010), Taylor and leaf PBZ, Induction of bulblets was obtained from 4.90 mM IBA Van Staden (2001a) 2,4-D, BA, 2-iP, Optimum shoot initiation was obtained from 1 mg/l:1 mg/l

Zeatin, mT, GA3, (NAA:BA). Root initiation was achieved from 1 mg/l IAA

GA4þ 7, ABA, MeJA, PAA

NR—Not Reported; Conservation status according to Raimondo et al. (2009). 2,4-D¼2,4-Dichlorophenoxy acetic acid; 2-iP¼N6-Isopentenyladenine; BA¼Benzyladenine; GA3¼Gibberellic acid; GA4þ7¼GA4 and GA7 gibberellin mixture; IAA¼Indole acetic acid; IBA¼Indole butyric acid; MeJa¼Methyl jasmonate; mT¼meta-Topolin; MS¼Murashige and Skoog (1962) media; NAA¼α-Naphthalene acetic acid; PAA ¼Phenylacetic acid; PBZ¼Paclobutrazol.

renal failure are some of the symptoms caused by Eucomis pois- investigation. Such valuable information will be vital in traditional oning in humans. Poisoning may be due to the hemolytic toxin medicine as well as from scientific and commercialization contained in the plants (Mander et al., 1995). Although the plant is perspectives. a member of the Hyacinthaceae family, cardiac glycosides which are widely distributed in the family have not been detected in Eucomis species (Watt and Breyer-Brandwijk, 1962). As recently 6. Conservation status reviewed by Koorbanally et al. (2006a), there is increasing evi- dence on the toxicity of crude extracts and isolated compounds Although legislation protecting medicinal plants such as Euco- from a number of Eucomis species. Nevertheless, the limited (if mis has been established (Mander et al., 1995), harvesting of plants any) information on safety evaluation remains worrisome. There- from their natural habitats remain unabated (Taylor and Van fore, it will be pertinent to subject the various Eucomis species, Staden, 2001a). Amongst the Eucomis species, Eucomis autumnalis especially the ones demonstrating potent bioactivity, to a is the most widely used species (Watt and Breyer-Brandwijk, 1962; sequence of toxicological and mutagenic (both in vitro and Hutchings, 1989; Roberts, 1990; Hutchings et al., 1996). As indi- in vivo) evaluation. The effect of mode of administration, dosage cated by street traders, Eucomis autumnalis and Eucomis bicolor are and age as well as gender on incidence of toxicity requires amongst the most popular and widely traded species in Durban, 52 N.A. Masondo et al. / Journal of Ethnopharmacology 151 (2014) 44–53

South Africa (Cunningham, 1988; Mander, 1998). According to No doubt, these protocols are valuable for the mass propagation of Cunningham (1990), Eucomis autumnalis is the second most Eucomis species. Therefore, it will be pertinent to provide protocols widely traded species in KwaZulu-Natal, South Africa. Subse- for the other Eucomis species which have not received much quently, Eucomis autumnalis populations have been described as attention. Using the new group of cytokinin (topolins) which have declining (Mander, 1997). Recently, Eucomis autumnalis was listed been demonstrated to be valuable in micropropagation (Aremu as endangered by the Conservation of Nature and Natural Resource et al., 2012), efforts aimed at improving shoot proliferation in (IUCN) (Victor, 2000). Increased harvesting of Eucomis autumnalis Eucomis autumnalis subspecies autumnalis are currently on-going. has significantly contributed to the shortage of the species in informal medicinal markets in South Africa (Govender et al., 2001). Table 5 provides a summary of the conservational status of 7. Conclusions members of the genus Eucomis. Apart from the medicinal uses of Eucomis, potential of the species as an ornamental/horticultural The continuous exploitation of the genus in southern Africa and plant due to their ‘eye-catching’ flowers has added more strain on especially in South Africa is an indication of its pharmacological wild populations. Therefore, the propagation of Eucomis species potential. In view of the potential of the genus in anti-inflam- especially the widely utilized Eucomis autumnalis and the vulner- matory therapy, the high COX inhibitory activity of crude extracts able Eucomis vandermerwei remains of outmost importance for the of active species such as Eucomis autumnalis subspecies autumnalis conservation of members of the genus. and Eucomis bicolor in vitro should be further investigated through in vivo bioassays. In order to achieve any pharmaceutical potential, 6.1. Conventional propagation it will be necessary to eliminate false positive results by removal of compounds such as polyphenols, saponins and fatty acids in Eucomis species can be propagated conventionally as offsets plant extracts which are known to affect enzyme-based bioassays. and by seeds. However, the propagation process is very slow In terms of the antimicrobial potential, the genus Eucomis is (Verdoorn, 1973). On average, they reach maturity within approxi- reported to be effective against Bacillus subtilis, Escherichia coli mately 3 to 4 years, and a bulb diameter of around 12 cm is and Staphylococcus aureus as well as several plant fungal strains. required for floral initiation. Generally, the plant requires sunny or In fact, crude extracts of Eucomis species were more potent than partially shaded areas/habitats for improved growth. Seed propa- the positive control against plant pathogens (Sclerotium rolfsii, gation is an effective method in conserving Eucomis species but Rhizoctonia solani and Pythium ultimum). Hence, Eucomis species the knowledge on seed biology is very limited. A germination rate can be potentially useful in the agricultural sector as a fungicide or of approximately 65% had been obtained (Diederichs et al., 2002). biocontrol agent. Based on the inadequate evidence on the general Seed germination of Eucomis autumnalis subspecies autumnalis safety of members of the genus, the need for detailed toxicological was found to be enhanced by dark conditions to obtain 100% and mutagenic evaluation is recommended. From a conservational germination (Kulkarni et al., 2006). perspective, renewed efforts aimed at cultivation and application In order to improve and gain better insight into growing of micropropagation techniques will definitely help alleviate the Eucomis, Ndhlala et al. (2012) investigated the essential environ- declining status of many of the heavily harvested species, parti- mental conditions required for the growth of Eucomis autumnalis cularly Eucomis autumnalis. Consequently, more studies geared seedlings. The level of light exposure (50%) was vital for Eucomis towards understanding the basic requirements of improving their autumnalis seedling growth. In terms of temperature, 25 1C and cultivation and micropropagation processes are encouraged. alternating 30/15 1C were the most preferred for seedling growth. Application of smoke-water (1:250 v/v) significantly enhanced Acknowledgments seedling growth.

6.2. Micropropagation of Eucomis Drs A.O. Aremu, M. Moyo, S.O. Amoo and W.A. Stirk are thanked for their valuable suggestions on the manuscript. Financial support Although efforts geared towards improving conventional pro- from the National Research Foundation, Pretoria and the Univer- pagation is commendable, the slow growth of Eucomis species sity of KwaZulu-Natal (Pietermaritzburg), South Africa is which may take as long as 3 to 4 years for bulb maturation appreciated. remains a major concern. Inevitably, the application of valuable techniques such as micropropagation (known for its numerous References benefits) has been embraced. The technique is useful for the conservation of species by increasing the production turn-over Amschler, G., Frahm, A.W., Hatzelmann, A., Kilian, U., Muller-Doblies, D., Muller- rate and reducing the growth duration significantly. The success of Doblies, U., 1996. Spirocyclic nortriterpenes from the bulbs of – fl viridifolia. Planta Med. 62, 534 539. hybridization in Eucomis owers has been partly attributed to the Aremu, A.O., Bairu, M.W., Doležal, K., Finnie, J.F., Van Staden, J., 2012. Topolins: a fact that the hybrids can be further propagated and remain true- panacea to plant tissue culture challenges? Plant Cell Tissue Organ Culture 108, to-type using subsequent micropropagation methods. 1–16. Ault, J.R., 1995. In vitro propagation of Eucomis autumnalis, E. comosa, and E. As evidence of the success and increasing application of zambesiaca by twin-scaling. HortScience 30, 1441–1442. micropropagation for the genus Eucomis, Table 5 shows members Bisi-Johnson, M.A., Obi, L.C., Kambizi, L., Nkomo, M., 2010. A survey of indigenous of the genus that have been tissue cultured. For instance, the herbal diarrhoeal remedies of O.R. Tambo district, Eastern Cape Province, South – micropropagation of Eucomis autumnalis and Eucomis zambesiaca Africa. Afr. J. Biotechnol. 9, 1245 1254. Bisi-Johnson, M.A., Obi, C.L., Hattori, T., Oshima, Y., Li, S., Kambizi, L., Eloff, J.N., using twin-scales has been successfully conducted (Ault, 1995). Vasaikar, S.D., 2011. Evaluation of antibacterial and anticancer activities of some McCartan and Van Staden (1995) focused on the tissue culture of South African medicinal plants. BMC Complement Altern. Med. 11, 14. Eucomis pole-evansii with the use of seedling explants while Bryan, J.E., 1989. Bulbs volume I A-H. Timber Press, Portland, Oregon. Cheesman, L., Finnie, J.F., Van Staden, J., 2010. Eucomis zambesiaca Baker: factors McCartan et al. (1999) devised a protocol for Eucomis vandermer- affecting in vitro bulblet induction. S. Afr. J. Bot. 76, 543–549. wei, one of the most vulnerable species within the genus. In vitro Clarkson, C., Maharaj, V.J., Crouch, N.R., Grace, O.M., Pillay, P., Matsabisa, M.G., propagation of 11 Eucomis species was conducted by Taylor and Bhagwandin, N., Smith, P.J., Folb, P.I., 2004. In vitro antiplasmodial activity of medicinal plants native to or naturalised in South Africa. J. Ethnopharmacol. 92, Van Staden (2001a). An improved protocol for the propagation 177–191. of Eucomis zambesiaca was described by Cheesman et al. (2010). Compton, J., 1990. Eucomis L’Heritier. Plantsman 12, 129–139. N.A. Masondo et al. / Journal of Ethnopharmacology 151 (2014) 44–53 53

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