Crinum bulbispermum (Burm. f.) Milne-Redh. & Schweick. is a deciduous bulbous plant which belongs to the Amaryllidaceae family. Crinum bulbispermum was initially described as Amaryllis bulbispermum Burm. f. and then mistakenly identified as Amaryllis longifolia L. which is now Cybistetes longifolia (L.) Milne-Redh. & Schweick. [1-3]. However, Milne-Redhead and Schweickerdt [2] simplified the matter in their revision of the genus Ammocharis by suggesting the new combination, C. bulbispermum, based on A. bulbispermum Burm. [3]. Crinum bulbispermum is a perennial herb growing to about 1 m tall, with a very large bulb, 10-11 cm in diameter covered with bases of older leaves and vermiform roots at the base [4]. Crinum bulbispermum has many, thick, stout, simple and glaucous green leaves, measuring 78-92 cm long, 6-6.7 cm broad, oblong to linear in shape, acuminate, flat and shining, margin slightly scabrous and undulate [3]. The peduncle is 50-90 cm long and has an umbel with between 6 to 16 flowers, which are white in colour with a dark red keel or entirely suffused with red; the stamens are declinate, white or suffused with pink and the style deep pink in the upper portion with a light brown stigma [4]. Crinum bulbispermum occurs naturally along rivers and streams or in damp depressions in black clay or sandy soils in Lesotho, South Africa and Swaziland [4].

The plant is cultivated throughout the world for its beautiful and trumpet or bell-shaped flowers. Like most medicinal plants in Southern Africa, C. bulbispermum is collected from the wild. The unsustainable harvesting of C. bulbispermum as herbal medicine and ornamental plant is threatening its continued existence. Although C. bulbispermum is widespread in South Africa, its population is declining due to over-exploitation of its bulbs which are sold in the medicinal (muthi) markets in Durban [5,6], Johannesburg [6,7] and Polokwane [8] in South Africa. Raimondo et al [9] categorized C. bulbispermum as declining in South Africa based on the modified IUCN Red List Categories and Criteria version 3.1 of threatened species [10-12]. According to Victor and Keith [11] and von Staden et al [12], a species listed as Least Concern (LC) under the IUCN Red List Categories and Criteria version 3.1 [10] can additionally be categorized either as rare, critically rare or declining. The observed population decline of C. bulbispermum in South Africa [9] is due to over-exploitation as an ornamental plant, medicinal plant trade and popularity of the species in the medicinal plant (muthi) markets. It is within this context that the current study was carried out, aimed at comprehensively documenting the ethnobotany, phytochemistry and pharmacology of C. bulbispermum so as to highlight research gaps and provide a foundation for further investigations on the plant species.

Traditional medicinal usage

Crinum bulbispermum is a popular medicinal plant in both rural and urban communities in Southern Africa [6,7,13,14]. It is commonly known as Orange river lily in English in South Africa and it is also known by various vernacular and synonym names in different geographical regions in Lesotho, South Africa and Swaziland (see ). In South Africa, the roasted bulbs of C. bulbispermum are applied to aching joints, rheumatism, varicose veins and backache, kidney or bladder infections and are used as poultices for septic sores and abscesses [13]. The southern Sotho in South Africa use the leaves and sliced or crushed bulbs to make a strong brew for treating colds, coughs, and as an external application or wash for wounds, scrofula and haemorrhoids (). According to Roberts [13], the leaves are used to bind dressings in place and flowers are placed over swollen joints and sprains to reduce swelling. Unspecified parts are used as infusions during pregnancy to ensure easy delivery [15]. The bulbs of C. bulbispermum are used for colds and to stimulate breast milk supplies by the Sotho in Lesotho [16]. Several tribes in Lesotho, South Africa and Swaziland are reported to use the juice squeezed from the base of the leaves to cure earache [13,17]. Sometimes pieces of roasted bulb are placed behind the ear or over the ear to ease the pain. Roberts [13] reports that some tribes in South Africa make a brew of the leaves which they believe to be an effective treatment for malaria. This same brew is drunk by the Zulu in South Africa as a treatment for rheumatic fever (usually half a cup chopped leaves in one cup boiling water and strained after standing for five minutes). The Tswana in South Africa drink a brew of crushed leaf bases and stalks to increase the flow of urine in bladder and kidney infections. The sliced bulb is also warmed and applied over the kidneys to ease discomfort [13]. Hutchings [18] records the use of C. bulbispermum bulb as a Zulu, Xhosa and Sotho gynaecological remedy and charm. Crinum bulbispermum leaves are browsed by cattle in South Africa [13].

Phytochemistry

Various reports on the phytochemical screening of C. bulbispermum bulbs, flowers, flowering stalks, leaves and roots confirm the presence of isoquinoline alkaloids, flavonoids, sterols, aldehydes, acids, esters, alcohols, esters, amines and amides, fatty acids and their esters (). Alkaloids are considered the major bioactive components of C. bulbispermum which exhibits various pharmacological effects [24,32,36-41]. Six distinct and structurally diverse isoquinoline alkaloids characteristic of the Amaryllidaceae family [42] have been isolated from C. bulbispermum, namely lycorine-type alkaloid (represented by eight alkaloids, alkaloids 1-8); galanthamine-type alkaloid represented by galanthamine 9; cherylline-type alkaloid represented by cherylline 10; crinine-type alkaloid (represented by 29 alkaloids, alkaloids 11-39), tazettine-type alkaloid (represented by 8α-ethoxyprecriwelline39, N-desmethyl-8α-ethoxypretazettine 40 and N-desmethyl-8β-ethoxypretazettine 41) and two minor alkaloids namely augustamine 42 and trisphaeridine 43 (). Non-alkaloid compounds isolated from C. bulbispermum bulbs, flowers and leaves are flavonoids represented by 15 compounds (compounds 44-58); sterols represented by dihydrositosterol 59 and stigmasterol 60; aldehydes, acids and esters represented by 4,5-methylenedioxy-4'-hydroxy-2-aldehyde-(1,1'-biphenyl) 61 and ρ-hydroxybenzene acetic acid ethyl ester 62; alcohols, esters, amines and amides represented by β-(3,4-dimethoxyphenyl)-α,β-ethanediol 63 and choline 64; and fatty acids and their esters represented by nine compounds, compounds 65-73 (see ). Although very little pharmacological evaluation of non-alkaloid compounds isolated from C. bulbispermum has been done to date, flavonoids are known to exhibit antioxidant activity, free radical scavenging capacity, coronary heart disease prevention, hepatoprotective, anti-inflammatory, anticancer and antiviral activities [43]. While phytosterols are known to have bioactive prevention properties such as lowering of cholesterol levels [44] and cancer prevention [45] properties. Previous research by Li et al [46] showed that unsaturated fatty acids have antifungal potencies, low toxicities and good pharmaceutical properties.

Pharmacological activities

A number of pharmacological activities of C. bulbispermum have been reported in literature justifying some of its ethnomedicinal uses. Some of the listed pharmacological activities may not relate directly to the ethnomedicinal uses of C. bulbispermum, but may provide some insight into its potential therapeutic value and bioactive properties. A wide range of biological activities have been reported including antimicrobial [36], antinociceptive [37], antiplasmodial [38], antioxidant [37,39], cytotoxicity and anti-apoptotic [24,37,40,41] as well as effects on the central nervous system (CNS) [37].

Effects on the central nervous system (NCS)

In recent years, members of the Amaryllidaceae family have been shown to contain alkaloids with promising acetylcholinesterace (AChE) inhibiting properties [47,48]. Adewusi and Steenkamp [39] found ethyl acetate extracts of C. bulbispermum bulbs and roots as well as methanol bulb extract to have some level of inhibitory activity against AChE with IC₅₀ values of 0.0021 ± 0.007, 0.0393 ± 0.014 and 0.0148 ± 0.039 mg/mL, respectively.

In an earlier research, orally administered aqueous leaf extract of C. bulbispermum doses of 1, 1.5 and 3 g/kg showed central inhibitory activity and markedly impaired the four parameters of rat hold-board test indicating its sedative properties [37]. The ability of C. bulbispermum to inhibit acetylcholinesterase may be ascribed to several alkaloids which have been isolated from the plant species () indicating its potential for use in treatment of neurodegenerative diseases. Recently, the alkaloid galanthamine 9 isolated from C. bulbispermum () was approved in the United States, many European countries and many Asian countries for the treatment of Alzheimer’s disease (AD) [49]. Alzheimer’s disease is characterized by a progressive impairment of cognitive functions including loss of memory and the inability to perform basic daily life activities [48]. Based on the cholinergic hypothesis, these symptoms are the results of the reduction in brain acetylcholine activity due to the catabolism of acetylcholine by ACHE [48]. Other alkaloids isolated from C. bulbispermum which have been screened for AChE inhibition activity include crinine 11 which exhibited inhibitory activity with IC₅₀ of 461 μM, crinamine 28 (IC₅₀ = 300 μM and lycorine 1 (IC₅₀ = 213 μM) [48].

Antioxidant activity

A methanol bulb extract of C. bulbispermum showed some radical scavenging activity in ABTS assays with IC₅₀ value of 0.0685 ± 0.041 mg/mL [39]. The total phenolic, flavonol and flavonoid contents of C. bulbispermum roots were relatively high for both solvents tested with total phenol of 202.38 ± 0.50 mg tannic acid/g, flavonol (20.79 ± 0.10 mg quercetin/g) and flavonoid (9.18 ± 0.50 mg quercetin/g) [39]. The levels of these phenolic compounds are an indication of the potential antioxidant activity of the plant extracts as phenolic compounds are well known as radical scavengers, metal chelators, reducing agents, hydrogen donors and singlet oxygen quenchers [50,51]. Additionally, the leaf extracts of C. bulbispermum showed modest antioxidant activity with EC₅₀ value of 203.76 µg/mL which was assessed by the thiobarbituric acid reactive substances assay [37]. These findings support the traditional use of the plant species for treating neurological disorders especially those involving cholinesterase mechanism and reactive oxygen species.

Antinociceptive activity

Ratnasooriya et al [37] evaluated the antinociceptive activity of C. bulbispermum leaves using three models of nociception namely tail flick, hot plate and formalin tests in male rats. The results showed that the leaf extracts had marked antinociceptive potential, particularly, when evaluated in the formalin test. According to Ratnasooriya et al [37], the obtained results suggest that the antinociception is mediated both spinally and supraspinally and is effective against phasic and continuous non-inflammatory or inflammatory pain. Ratnasooriya et al [37] attributed the antinociception of the leaf extract of C. bulbispermum to the results of opioid mechanisms, sedation and antioxidant activities of the species. These results support the traditional use of the species in various inflammatory ailments and diseases ranging from microbial infection to injury that result in swelling, cell injury and death.

Antiplasmodial activity

Van Dyk et al [38] screened C. bulbispermum for antiplasmodial activity using the [3H]-hypoxanthine incorporation assay against the chloroquine-resistant Plasmodium falciparum. Extracts of C. bulbispermum had IC₅₀ values ≤ 1 µg/mL with the ethyl acetate extracts of the roots and bulbs having values comparable to chloroquine (0.04 µg/mL). Van Dyk et al [38] identified lycorine 1 as a potent antiplasmodial compound with an IC₅₀ value of 0.03 µg/mL against the chloroquine-resistant strain (FCR-3) of P. falciparum, which is comparable to the activity of the crude extract and chloroquine. The most promising extract was the ethyl acetate bulb extract of C. bulbispermum with an IC₅₀ value of 0.08 µg/mL and a security index of 2203.13. Likhitwitaywuid et al [52] reported an IC₅₀ value of 0.3 μg/mL for lycorine 1 isolated from C. amabile against the chloroquine-resistant strain (W-2).

Antimicrobial activity

Griffiths [36] assessed antimicrobial activity of C. bulbispermum using the direct plate method and minimum inhibitory concentration values were determined. The best activity was observed for the alkaloid lycorine 1 against Bacillussubtilis [36]. One of the most common ethnomedicinal uses of C. bulbispermum is in the treatment of a wide range of infectious diseases caused by microorganisms such diseases or ailments include symptoms such as sores [17] and wounds [13]. Previous research showed that some of the alkaloids that have been isolated from C. bulbispermum have antibacterial activity. For example, the alkaloid crinamine 28 is known to have antibacterial activity, as it showed some strong activity against Bacillus subtilis and Staphylococcus aureus [53].

Cytotoxicity and anti-apoptotic activity

Seoposengwe et al [41] evaluated the cytoprotective potential of C. bulbispermum, after induction of toxicity using rotenone, in SH-SY5Y neuroblastoma cells. Rotenone reduced intracellular reactive oxygen species (ROS) levels after 24 h exposure. Pre-treating cells with C. bulbispermum extracts reversed the effects of rotenone on intracellular ROS levels. Rotenone exposure also decreased intracellular glutathione levels, which was counteracted by pre-treatment with any one of the extracts. MMP was reduced by rotenone, which was neutralized by pre-treatment with C. bulbispermum ethyl acetate extract. All extracts inhibited rotenone-induced activation of caspase-3. Crinum bulbispermum demonstrated anti-apoptotic activity and restored intracellular glutathione content following rotenone treatment, suggesting that they may possess neuroprotective properties.

Ratnasooriya et al [37] evaluated the sub-chronic toxicity of the aqueous leaf extract of C. bulbispermum. The extract induced mild to moderate toxicity in rats which developed diarrhoea and postural abnormalities on the second day, and two rats died by the fourth day. Liver and renal toxicities (increase of serum SGOT, SGPT, creatinine and urea) were also reported and the authors attributed this toxicity to the lycorine- and crinine-types of alkaloids present in C. bulbispermum (see ). According to van Wyk et al [54] the major toxic compound in C. bulbispermum is the alkaloid crinamine 28, which is regarded as highly lethal with an oral lethal dose LD₅₀ at a concentration of 10 mg/kg body weight in dogs. Crinamine 28 is regarded as a powerful transient hypotensive in dogs and also shows respiratory depressant activity [55]. Similarly, Aboul-Ela et al [24] tested cytotoxicity of C. bulbispermum bulbs using the brine shrimp bioassay. The most effective one were the butanol fraction of the acidic extract of the non-flowering bulbs with LD₅₀ of 63.1 μg/mL followed by the ether fraction of the alkaline extract of the flowering bulbs with LD₅₀ of 73 μg/mL.

Van Dyk et al [38] evaluated the cytotoxicity of C. bulbispermum compounds as determined by in vitro cellular toxicity assay, reporting the IC₅₀ value of 445.47 μg/mL for lycorine 1 against human kidney epithelial cells and toxicity index of > 15 000. Other researchers like Campbell et al [56] evaluated cytotoxicity activities of alkaloids associated with C. bulbispermum reporting IC₅₀ values of 0.6 and 0.7 μg/mL for lycorine 1 against the strains D-10 and FAC8, respectively. Likhitwitaywuid et al [52] reported IC₅₀ values for ranging from 0.3 to 1.6 μg/mL against a series of human cancer cells. The anti-cancer activity of lycorine 1 was also reported by Li et al [57] as an effect due to the apoptosis inducing effect of lycorine 1 and it has been found to inhibit protein synthesis. Abd El Hafiz et al [32] examined the constituents of C. bulbispermum for activity against human leukemic Molt 4 cells. Of the flavan, 4'-hydroxy-7-methoxyflavan 44 and three crinanes namely powelline 36, crinine-6-α-hydroxy 31 and buphanisine-6-α-hydroxy 23 tested, only buphanisine-6-α-hydroxy 23 and 4'-hydroxy-7-methoxyflavan 44 were moderately active, causing a steady decline (up to about 20 %) in the viability of leukemia cells over the three-day treatment period at a dosage of 71 µg/mL.

Seoposengwe et al [41] found the ethyl acetate extract of C. bulbispermum to have the least cytotoxic with an LC₅₀ value of > 100 μg/mL. Contrary to this, the methanol extract produced an LC₅₀ value of 46.18 ± 0.91 μg/mL. Adewusi et al [40] evaluated cytotoxicity of C. bulbispermum ethanol extracts against SH-SY5Y (human neuroblastoma) cells as well as toxically induced with Aβ, using the MTT and neutral red uptake assays. The extracts of the root and bulb of C. bulbispermum were the most toxic with IC₅₀ values < 50 μg/mL in both assays. However, despite the observed toxicity, the roots and bulbs of C. bulbispermum still reduced the cell death induced by Aβ at less toxic doses. These results show that C. bulbispermum may contain several alkaloids with possible neuroprotective activities.

The widespread usage of C. bulbispermum as an ornamental plant and in traditional medicine resulting in negative impact on wild populations calls for conservation strategies and mechanisms for sustainable utilization of the species. The estimated declining rate of C. bulbispermum [9] is based on the decreasing population numbers at known habitat sites and reduction in the bulb sizes of the species sold in medicinal muthi markets [7]. Therefore, propagation protocols for C. bulbispermum should be developed as an alternative and viable means to provide sufficient plants to meet the demand for ornamental and medicinal purposes and at the same time protecting the natural populations. There is also need for comparative evaluation of the phytochemistry and biological activities of the various plant parts in an attempt to suggest plant part substitution as a means of conserving this highly collected plant species. If phytochemical and biological analyses prove that the leaves, flower stalks, flowers and fruits have comparable properties and activities to those of the bulbs and roots, then plant part substitution can be used to curtail the destructive harvesting of bulbs that has led to a continuous decimation of the wild populations.

The present review summarizes ethnomedicinal uses, phytochemistry, biological activities and cytotoxicity of different extracts and compounds of C. bulbispermum. Crinum bulbispermum has been traditionally used as herbal medicine throughout its distributional range in southern Africa, used for the treatment of common diseases and ailments like colds, cough, earache, haemorrhoids, malaria, wounds and others medical complications like gynaecological, kidney and bladder problems. Recent research on C. bulbispermum focused primarily on evaluating the antimicrobial, antioxidant, antiplasimodial, cytotoxicity activities of the species as well as the effects on the central nervous system. Alkaloids appear to be the major ingredients in C. bulbispermum bulbs, flowers, flower stalks, leaves and roots and these compounds appear to be responsible for the pharmacological properties of the species. Other important phytochemical constituents isolated from C. bulbispermum bulbs, flowers and leaves are flavonoids, sterols, aldehydes, acids and esters, alcohols, esters, amines, amides and fatty acids and their esters. Surprisingly, there is no systematic data linking the ethnomedicinal uses of C. bulbispermum to the phytochemical and pharmacological properties of these non-alkaloid compounds. Future studies should therefore, try to establish a link between the phytochemical and pharmacological properties of the non-alkaloid compounds and the ethnomedicinal uses of C. bulbispermum.

Although contemporary research involving C. bulbispermum is promising, it is too preliminary and sometimes too general to be used to explain and support its ethnomedicinal uses. Most of the mentioned phytochemical constituents and pharmacological studies have provided some suggestive scientific evidence for the various ethnomedicinal uses of C. bulbispermum in the treatment of parasitic diseases such as control and management of malaria, inflammatory ailments and wounds; there is need for extensive phytochemical, pharmacological, preclinical and clinical research. There is yet not enough systematic data regarding the pharmacokinetics and clinical research of C. bulbispermum products and compounds. There are very few to nil experimental animal studies, randomized clinical trials and target-organ toxicity studies involving C. bulbispermum and its derivatives that have been carried out so far. Therefore, studies should identify the bioactive components, details of the molecular modes or mechanisms of action, pharmacokinetics and physiological pathways for specific bioactives of C. bulbispermum. Future studies should include the identification of any side effects and/or toxicity.