Journal of Ethnopharmacology 150 (2013) 818–827 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep Review Hypoxis (Hypoxidaceae) in African traditional medicine Bhekumthetho Ncube, Ashwell R. Ndhlala, Ambrose Okem, 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 art ic l e i nf o a b s t r a c t Article history: Received 22 August 2013 Received in revised form 16 October 2013 Accepted 19 October 2013 Available online 30 October 2013 Ethnopharmacological relevance: Hypoxis species (Hypoxidaceae) are popular medicinal plants used in the African traditional medicine to treat numerous ailments and are reported to have a wide spectrum of pharmacological properties. In this paper we reviewed and evaluated the traditional uses, pharmacological, phytochemistry and toxicity aspects of the genus Hypoxis. Potential medicinal prospects and possible knowledge gaps and pitfalls are discussed. Materials and methods: A comprehensive and systematic review of literature on Hypoxis species was done using numerous resources such as books and scientific databases that include Pubmed, Scopus, Scirus, Google scholar, Web of Science and others. Information on the various pharmacological and chemical properties of Hypoxis extracts was critically analysed and discussed under the various topics. Results: The literature indicated a broad range of uses, pharmacological and toxicological properties of different Hypoxis species extracts and their relevance to African healthcare systems. Several compounds, mostly glucosides, sterols and sterolins, have been isolated, identified and tested in various in vitro and in vivo models as well as in a couple of clinical trials with a lot of promising prospects reported in some studies. Conclusion: A critical analysis of the available literature and studies identifies positive potential for the future use of Hypoxis species in both traditional and modern medicine and concurrently so with possible pitfalls and research gaps in current knowledge. An integrated and holistic approach to addressing research issues, particularly toxicology aspects could be more effectively applied and incorporate conservation strategies for the species. & 2013 Elsevier Ireland Ltd. All rights reserved. Keywords: African traditional medicine Hypoxis Hypoxoside Pharmacology Phytochemistry Toxicology Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819 Medicinal uses of Hypoxis species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819 2.1. Pharmacological activity of Hypoxis extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820 2.1.1. Antimicrobial and antiviral activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820 2.1.2. Anti-inflammatory and anti-diabetic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 821 2.1.3. Antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 822 2.1.4. Anticancer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 822 2.1.5. Cardiovascular and anticonvulsant activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 822 2.2. Phytochemistry and related bioactivities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823 3. Safety and toxicity issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 824 4. Future prospects and potential pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 824 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826 Abbreviations: ATM, African traditional medicine; COX, cyclooxygenase; FRAP, ferric reducing ability of plasma; GABAA, gamma-aminobutyric acid; HIV/AIDS, Human immunodeficiency virus/acquired immunodeficiency syndrome; LDL, low-density lipoprotein; NGO, Non-governmental organisation; NO, nitric oxide; PBMC, peripheral blood mononuclear cells; ROS, reactive oxygen species n Corresponding author. Tel.: þ 27 33 2605130. E-mail address: rcpgd@ukzn.ac.za (J. Van Staden). 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.10.032 B. Ncube et al. / Journal of Ethnopharmacology 150 (2013) 818–827 1. Introduction The rich and diverse floral landscape of the African continent forms part of the medicinal wealth that ensures the primary healthcare and livelihoods of millions of the African population. From an African Traditional Medicine (ATM) context, although animals form an integral component of the holistic approach to the treatment of a range of ailments and conditions (Cunningham, 1993; Bodeker, 2004), plants are regarded as the basis of this medical practice (Gurib-Fakim, 2006). A diverse range of medicinal plants, mostly harnessed from the indigenous flora, are used as herbal remedies and administered in various forms and play a crucial role in the healthcare of millions in most developing communities. The low socio-economic standing of the large, predominantly rural African population, the deep-rooted cultural beliefs and values coupled with the high cost and inaccessibility of the parallel medical systems are believed to be the major drivers for the majority's option for ATM. Be that as it may, and notwithstanding the various perceptions associated with ATM, the growing recognition of its value and importance continue to provide sound solutions to a number of ailments on a global perspective. In any ethno-based medicinal system, certain plant species tend to find more medicinal/ethnopharmacology usage than others in a number of ways and to treat a range of ailments. Such multiple usages create demand that generate a species-specific trade network that can extend even across international boundaries. Some members of the genus Hypoxis in the family Hypoxidaceae are one such group of plants that are popularly used particularly in southern Africa. Hypoxis is an ingredient to a number of herbal remedies. The genus is used by different African ethnic groups to cure various conditions and disorders. A number of literature contributions on African medicinal plants, document a diverse range of medicinal usage of Hypoxis extracts, ranging from the healing of septic sores, alleviation of headaches, dizziness, as purgatives and ascarifuge to the healing of testicular tumours, prostate hypertrophy, cancer, HIV, nervous disorders and urinary infections (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). Many more medicinal uses for the species continue to emerge regularly. Although some species show close resemblance in morphological features leading to misidentification during gathering and use, one species in particular, Hypoxis hemerocallidea Fisch. & C.A. Mey is a very popular herbal remedy in South Africa. In a survey carried out in the Eastern Cape Province, the species ranked top among the frequently traded and highly priced medicinal plants in the area, with Hypoxis filifolia also falling within the top 60 (Dold and Cocks, 2002). This also implies an obvious exploitation of the species in the wild. Following the isolation and elucidation of a phytosterol diglucoside, hypoxoside (Drewes et al., 1984) and 10 years later, the promising antitumour activity of its hydrolysed aglycone, rooperol (Albrecht et al., 1995a), the South African media stimulated a sudden surge in the public interest towards the use of this plant species. The public became enthused to experiment with Hypoxis preparations to cure a number of ailments. Hailed as ‘miracle muthi’ (Muthi is a Zulu term that refers to a herbal remedy) and ‘wonder potato’, Hypoxis has become a household name in South Africa and perhaps one of the best known ‘muthi’ plants in the country with the resultant controversies surrounding its use as an effective agent against HIV/AIDSrelated conditions and specific cancers (Drewes and Kahn, 2004). Among the people of Swaziland, Hypoxis species are often referred to as ‘zifozonke’, meaning: ‘the plant that can be used to treat many diseases’ (Amusan et al., 2007). The species, and mostly Hypoxis hemerocallidea and related species are sold in most herbal markets in southern Africa, and are an alleged component of numerous over-the-counter commercial medicinal preparation products 819 widely traded. Parallel to this, several pharmacological and phytochemical studies were undertaken using this and other Hypoxis species, employing both in vitro and in vivo models. Although not all Hypoxis species are extensively utilised in ATM, over 70 species are found in Africa. Given the huge medicinal demand for the species, and the morphological similarities between some species, it is highly likely that species are often substituted with each other for different medicinal uses. A case in point to reiterate this view is the close morphological resemblance between Hypoxis rigidula, Hypoxis hemerocallidea, Hypoxis obtusa, Hypoxis galpinii, Hypoxis colchicifolia and Hypoxis acuminata all of which are actively utilised in ATM. Some phytochemical studies also reveal similarities in the chemical profiles of most species (Boukes et al., 2008). In this review, we document the medicinal uses, medicinal products as well as pharmacological, phytochemical, clinical and toxicological studies on the Hypoxis species utilised in ATM. Future prospects and possible potential pitfalls on uses of the species for medicinal purposes are discussed. 2. Medicinal uses of Hypoxis species Hypoxis species are used by different indigenous population groups of Africa in a variety of ways and for various medicinal purposes as well as food. The wide range of medicinal uses include the treatment of intestinal parasites, urinary infections, infertility, vomiting, nausea, cough, palpitations, heart weakness, impotency, anxiety, insanity, lice, common cold, flu, wounds, arthritis, cancers, conditions related to HIV/AIDS, hypertension, diabetes, cancer, psoriasis, gastric and duodenal ulcers, tuberculosis, asthma, some central nervous system (CNS) disorders (including epilepsy and childhood convulsions), depression, laxative, vermifuge, burns, prostatitis, benign prostatic hyperplasia, prostate adenoma and many more (Phillip, 1917; Watt and Breyer-Brandwijk, 1962; Sibanda et al., 1990; Hutchings et al., 1996; Risa et al., 2004; Buwa and Van Staden, 2006; Ojewole, 2006; Koduru et al., 2007; Drewes et al., 2008). Weak infusions and decoctions of Hypoxis are used as convalescent and strengthening tonics for wasting diseases (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). Table 1 lists some of the different Hypoxis species used in ATM, region most used and their known chemical constituents. Hypoxis hemerocallidea is a medicinal plant that is used to treat almost all kinds of human and veterinary diseases in South Africa (Ojewole, 2006) and perhaps is one of the most researched medicinal plants in Africa. The medicinal value of Hypoxis hemerocallidea was established a long time ago particularly among the Zulu people of South Africa (Drewes et al., 2008). Traditionally, decoctions of this plant species are used for cardiac diseases, impotency, barrenness, intestinal parasites, cancer, headaches and testicular tumours (Drewes et al., 2008). Infusions of the corm are also used as emetics to treat dizziness, urinary tract infections and insanity (Hutchings et al., 1996). Decoctions of Hypoxis colchicifolia are used extensively to treat infertility and convulsions (Pooley, 1998; Risa et al., 2004). Botanical formulations based on Hypoxis hemerocallidea became popular as far back as 1967, when R.W. Liegenberg, an entrepreneur from Johannesburg initiated the use of Hypoxis phytosterols, β-sitosterol and its glucoside products. The product, marketed as Harzols is used for the amelioration of benign prostatic hypertrophy which gained wide acceptance in Germany (Bouic et al., 1996; Schulz et al., 2001; Drewes et al., 2008). Several other products containing phytosterols from Hypoxis have been popularised as human immune system boosters and today products of this nature are freely available as ‘over the counter’ (OTC) preparations (Drewes et al., 2008). Moducares is one example of such a product. It consists of capsules containing 20 mg of β-sitosterol and 0.2 mg of its glucoside as this particular ratio is 820 B. Ncube et al. / Journal of Ethnopharmacology 150 (2013) 818–827 Table 1 Traditional uses and chemical constituents of some Hypoxis species used in African traditional medicine. Species Area used Traditional uses Known chemical constituents References Hypoxis acuminata Barker Hypoxis angustifolia Lam. Swaziland Corms taken as medicinal food Geraniol acuminoside, hypoxoside Bredenkamp et al. (1989) Zimbabwe Corms eaten as a medicinal food either raw, boiled or roasted Nyasol, 1,3(5)-diphenyl-1pentene, hypoxoside, nyasoside, nyaside and mononyasine A and B. Sibanda et al. (1990) and Tredgold (1986) Swaziland Lesotho Used for making a good luck charms Mixed with fat to treat cracks on the teats of cows Corms taken as food Hypoxis argentea Harv. ex Baker Hypoxis gerrardii Barker Hypoxis hemerocallidea Fisch. & C.A. Mey South Africa Swaziland South Africa Southern Africa Corms boiled and use to treat stomach problems and dysentery Used as a cure for headaches, stomach ailments, dysentery, dizziness, burns, cancer, HIV, symptoms of benign prostrate hyperthrophy, diabetes, high blood pressure, pimples, wounds, skin rush, dermatitis, mental disorders and as a general tonic for good health. Used as a purgative Used against barrenness, bad dreams, heart weakness. Used as a snake trap, emetic, to treat nausea, insect bites, placed in food to destroy small vermin. Used as a purgative, diuretics and ascarifuge Used to treat abdominal pains, urinary diseases, infertility and as an aphrodisiac. Also used in mock bull fights Induces perspiration, used as a cough remedy and for treatment of various ailments Hypoxis colchicifolia Barker Southern Africa Hypoxis obtusa Busch Southern Africa Hypoxis nyasica Barker Whole of Africa Hypoxis multiceps Buchinger ex Baker Hypoxis obliqua Jacq. Hypoxis rigidula Barker Hypoxis interjecta Nel. Lesotho Swaziland Used as a charm against lighting South Africa Used to treat wounds Southern Africa Used to treat gall sickness in cattle and a variety of ailments in humans Used as a charm against lighting Whole of Africa regarded as ‘critical’ for the effectiveness of the product (Bouic, 2002). There are also numerous other commercially available formulations of Hypoxis extracts with various therapeutic claims (Table 2). 2.1. Pharmacological activity of Hypoxis extracts 2.1.1. Antimicrobial and antiviral activity A number of extracts from Hypoxis species have demonstrated antibacterial, antifungal and antiviral activities against numerous pathogenic strains associated with acute infections (Buwa and van Staden, 2006; Steenkamp et al., 2006). Evidence-based laboratory investigations indicate that aqueous and alcoholic extracts of Hypoxis hemerocallidea possess a number of promising pharmacological properties (Owira and Ojewole, 2009). Crude ethyl acetate extracts of Hypoxis hemerocallidea corms exhibited potent antimicrobial activities against Staphylococcus aureus and Enterococcus faecalis with MIC values of 0.31 and 0.63 mg/ml respectively using a microdilution assay (Katerere and Eloff, 2008). Following a similar test procedure, Ncube et al. (2011) reported water and lipophilic leaf and corm extracts of the Phillip (1917) and Watt and Breyer-Brandwijk (1962) Phytosterol glucosides (β-sitosterol), diglucoside hypoxoside, aglycone rooperol, sterols and sterolins Hutchings et al. (1996) and Watt and Breyer-Brandwijk (1962) Drewes et al. (1984), Hutchings et al. (1996), Katerere and Eloff (2008), Phillip (1917) and Watt and BreyerBrandwijk (1962) Haemanthine, Pentenye-bisglucosidophenol, hypoxoside Bryant (1966), Drewes and Liebenberg (1983), Hutchings et al. (1996), Pooley (1998), and Watt and Breyer-Brandwijk (1962) Hypoxoside, obtusaside Marini-Bettolo et al. (1982) and Nicoletti et al. (1992) Alkaloid, monoglucoside nyasicoside, hypoxoside, nyasol, mononyasine A and B, nyaside, nyasoside Nyasicoside, bisphenylpentanoid norlignans Galeffi et al. (1987), Marini-Bettolo et al. (1985), Messana et al. (1989), Nicoletti et al. (1992), and Watt and Breyer-Brandwijk (1962) Marini-Bettolo et al. (1991) and Phillip (1917) Watt and Breyer-Brandwijk (1962) Organic acids, phenolic compounds Interjectin, bisphenylpentanoid norlignans, hypoxoside, Obtusides A and B, tetramethylnyasicoside Watt and Breyer-Brandwijk (1962) and Whiten et al. (1991) Marini-Bettolo et al. (1991) same species to have good antibacterial and anticandidal activity in autumn and winter against Bacillus subtilis, Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus and Candida albicans. A remarkable complete inhibition of Escherichia coli growth at a concentration of 100 mg/ml was recorded from aqueous and methanolic extracts of this Hypoxis species (Steenkamp et al., 2006). When prepared as a tea, approximately 37 mg/ml of Hypoxis hemerocallidea extract is consumed three times a day (Steenkamp et al., 2006). Assuming total absorption, the authors extrapolate a serum concentration of 18.5 μg/ml. Compared with the MIC that caused total inhibition of Escherichia coli growth (62.5 μg/ml) by aqueous and ethanolic extracts, this concentration seem to be physiologically irrelevant. Excellent agglutination activity against Staphylococcus aureus and Bacillus subtilis (0.7 and 4.1 mg/ml) respectively was reported from lectin-like proteins extracted from Hypoxis hemerocallidea (Gaidamashvili and Van Staden, 2002). The active compound rooperol exhibited five times lower MIC against Escherichia coli and Staphylococcus aureus at 1.2 mM compared to neomycin as a positive control in a microdilution assay (Laporta et al., 2007). Furthermore, in a synthetic membrane model study, the compound promoted high membrane leakage in Escherichia coli but B. Ncube et al. / Journal of Ethnopharmacology 150 (2013) 818–827 821 Table 2 Examples of some herbal formulations involving Hypoxis corm extract constituents available in South Africa. Trade name Form Therapeutic claims Available at Composition Moducares Capsule Hypo-Plus Capsule Immune booster, for treating flu and infections, as well as allergies and painful autoimmune disorders such as rheumatoid arthritis As food supplement, energy booster and immunity modulator 20 mg of β-sitosterol and 0.2 mg of its glucoside and this particular ratio is regarded as ‘critical’ for the effectiveness of the product Contains a variety of amino acids, a selection of vitamins and Hypoxis extract African potato/ Hypoxis hemerocallidea Immuniser Capsule Used to boost the immune system and treatment of HIV/AIDS symptoms Health stores and Pharmacies Health stores and Pharmacies Health stores Capsule Immune system enhancer Pharmacies Herbal immune booster Medico herbs African Potato (Hypoxis hemerocallidea) Immuno active Down to earth Tincture Used to boost the immune system Pep stores Capsule Regulate immune system and reduce allergy symptoms Capsule Regulate immune system and reduce allergy symptoms Health stores Health stores Capsule Cream Used to boost the immune system Skin blemishes, rheumatoid arthritis, acne, antibacterial, antifungal Natural HIV treatment, immune booster 300 mg of dry extract of Hypoxis hemerocallidea, per capsule Undisclosed amounts of Hypoxis hemerocallidea and Aloe vera 500 mg of Hypoxis and 55 mg plant sterols per 20 mL Sterols and sterolins Dry extract of Hypoxis hemerocallidea 60  350 mg capsules Pharmacies Health stores Pharmacies and Health stores Immune booster, treats enlargements of prostate gland, urinary Pharmacies and Health tract infections, cancer and tumour growth and rheumatoid stores arthritis 500 mg of Hypoxis hemerocallidea. Also contain Aloe ferox Sterols Afrigetics™ Hypoxis Capsule African Potato Capsules (Hypoxis) – Phyto Green African potato extract – South Africa's miracle herb Stameta Capsule Tonic Used to boost the immune system and treatment of HIV/AIDS symptoms Health stores Sterols and sterolins Tonic Health stores Hypoxis rooperi extracts, Mentha piperital, Pimpinella anisum, Aloe (unspecified). Fortified with multivitamins (unspecified), calcium, magnesium, potassium, phosphorus and iron African potato Tonic Used for nervous disorders, skin conditions, boosts sexual performance, poor blood quality, high blood pressure. Chest, lung and kidney infections. Fever and flu. Heart problems, back pain, persistent tiredness. Menstrual pain, cleans out bile, bleeding gums, body sores. Strengthens bones and boosts the immune system Rheumatism, high blood pressure, gout and arthritis, cancer, TB, yuppi flu, psoriasis, eczema, varicose veins bad blood circulation, prostate problems Health stores Unspecified amount of Hypoxis rooperi extracts. Contains sterols and sterolins with a much stronger inhibition on Staphylococcus aureus. The active compound rooperol exhibited a high physiological membrane leakage in an Escherichia coli model containing synthetic membranes. Inhibition of bacterial growth is through disruption of the phospholipid/ water interface through the formation of gel-fluid like intermediate structures with isotropic motion in phosphatidyglycerol membranes at physiological pH (Laporta et al., 2007). From Hypoxis latifolia extracts, Buwa and Van Staden (2006) reported good antimicrobial activity against a number of bacterial and fungal strains. Aqueous and ethanolic extracts of Hypoxis sobolifera exhibited significant in vitro anti-HIV activity with (Z 50%) inhibition at 0.2 mg/ml against HIV-1 RT using HIV-1 reverse transcriptase assay (Klos et al., 2009). Pharmacokinetics studies demonstrated that concurrent use of Hypoxis obtusa extracts with antiretroviral drugs exhibited neither clinical manifestation nor alteration of the absorption characteristics of drugs (Gwaza et al., in press). On the other hand, Hypoxis hemerocallidea showed interference with the efflux of nevirapine across intestinal epithelial cells and potentially increase the bioavailability of this antiretroviral drug when taken concomitantly (Brown et al., 2008). 2.1.2. Anti-inflammatory and anti-diabetic activity Hypoxis products are well known remedies for the management and/or control of painful arthritic and inflammatory conditions as well as for adult-onset type-2 diabetes. Several studies have been carried Dry extract of Hypoxis hemerocallidea 60  300 mg capsule Dry extract of Hypoxis hemerocallidea 60  450 mg per capsule out to support these claims. At a dose of 50–800 mg/kg body weight, oral administrations of Hypoxis hemerocallidea methanol and aqueous corm extracts significantly inhibited egg albumin-induced acute inflammation as well as reduce blood glucose levels in both normal and streptozotocin-induced diabetic rats in a dose-dependent manner (Ojewole, 2006). Intraperitoneal injections of Hypoxis hemerocallidea extracts produced anti-nociceptive effects against chemicallyand thermally-induced nociceptive pain in mice (Ojewole, 2006). Although numerous studies demonstrated some inhibitory activity against COX-1 and COX-2 enzymes by ethanol and aqueous extracts of Hypoxis hemerocallidea in both in vitro (Jäger et al., 1996; Steenkamp et al., 2006; Ncube et al., 2012) and in vivo (Ojewole, 2006) models, neither of the same extracts produced a significant inhibition of the two enzymes in vitro even at concentrations as high as 0.5 mg/ml (Laporta et al., 2007). In contrast, rooperol showed a noticeable inhibitory effect on both enzymes, with a stronger effect on COX-2 than COX-1, yielding a COX-2/COX-1 IC50 ratio of 1.9 (Laporta et al., 2007). Lectin-like proteins purified from aqueous extracts of Hypoxis hemerocallidea inhibit COX-1 enzyme that mediates prostaglandin synthesis in vitro (Gaidamashvili and Van Staden, 2006). The antiinflammatory activity of Hypoxis extracts are suggested to be more likely taking place via the inhibition of the prostaglandins, 5-lipoxygenase pathway and other inflammatory mediators such as antioxidant activity which in turn, inhibits COX enzymes (Van der Merwe et al., 1993; Feng et al., 1995; Guzdek et al., 1996; Steenkamp et al., 2006). 822 B. Ncube et al. / Journal of Ethnopharmacology 150 (2013) 818–827 Hypoglycaemic effects of Hypoxis hemerocallidea aqueous extracts were investigated on fasted normal and diabetic rats, with a dose-dependent (100–800 mg/kg) moderate to high response being recorded against insulin (5 mU/kg) and glibenclamide (5 mg/kg) as positive controls (Mahomed and Ojewole, 2003; Ojewole, 2006). At a dose of 800 mg/kg, the extract led to 30.2% and 48.5% reductions in the blood glucose concentrations of fasted normal and streptozotocin-treated diabetic rats, respectively (Mahomed and Ojewole, 2003). Drewes et al. (2008) concluded that the extracts could likely induce hypoglycaemia by stimulating insulin release thereby enhancing the cellular uptake and utilisation of glucose within the animal. 2.1.3. Antioxidant activity The inhibitions of free radicals leading to degenerative disorders by antioxidant agents have been explored for the prevention or alleviation of ageing (Drewes et al., 2008). The aqueous and ethanolic extracts of Hypoxis hemerocallidea demonstrated good antioxidant properties through hydroxyl scavenging ability (Steenkamp et al., 2006; Nair and Kanfer, 2007). In vitro studies indicate that aqueous extracts of Hypoxis hemerocallidea have good ability to scavenge free radicals (hydroxyl ions) (Mahomed and Ojewole, 2003). Rooperol significantly increased reactive oxygen species (ROS) and nitric oxide (NO) production, and phagocytosis in undifferentiated and/or differentiated human promonocytic U937 leukaemia cells than its positive control ascorbic acid (Boukes and Van de Venter, 2012). Using the ferric reducing ability of plasma (FRAP) assay, the authors later confirmed the antioxidant capacity of rooperol, results of which were consistent with those of previous studies (Van der Merwe et al., 1993; Guzdek et al., 1996; Mahomed and Ojewole, 2003; Steenkamp et al., 2006; Laporta et al., 2007). In a separate but related study, rooperol and Hypoxis hemerocallidea aqueous extracts reduced quinolinic acidinduced lipid peroxidation in rat liver homogenates and significantly scavenged the superoxide anion at pharmacological doses (Nair et al., 2007). At similar concentrations (8, 16 and 32 mg/ml) rooperol demonstrated significantly greater ferric reducing activity than ascorbic acid (Nair et al., 2007) while Boukes and Van de Venter (2012) reported similar results at a concentration of 20 mg/ml. Using albino rats, water decoction of Hypoxis corms (10 mg/kg) exhibited good antioxidant activity by inducing protection against oxidative stress generated by chloroquine in a dose dependant manner (Chaturvedi and Mwape, 2011). The bioactive compound rooperol isolated from Hypoxis hemerocallidea has a unique structural similarity to nordihydroguaiaretic acid which is a strong antioxidant, a phenomenon that could explain the antioxidant properties of these extracts (Van der Merwe et al., 1993; Boukes and Van de Venter, 2012). Using platelet microsomes, rooperol and nordihydroguaiaretic acid gave comparable results in the inhibition of leukotriene synthesis in the polymorphonuclear leucocyte and prostaglandin synthesis as well as stimulate the oxidation of haemoglobin to a greater extent (Van der Merwe et al., 1993; Coetzee et al., 1996). 2.1.4. Anticancer activity One of the prominent uses of Hypoxis extracts in traditional medicine is against cancer. To test the significance of these purported claims, Steenkamp and Gouws (2006) found that aqueous extracts of Hypoxis hemerocallidea at 50 mg/ml significantly stimulated DU-145 prostate cancer cell growth more than cisplatin, a known anti-tumour agent. Albrecht et al. (1995b) demonstrated that sitosterols from Hypoxis hemerocallidea have good in vitro activities by stimulating lymphocyte proliferative responses towards phytohaemoglutinin at very low concentrations, making these compounds essential nutrients to be taken on a daily basis for the optimal functioning of the immune system. In vitro studies on the compound hypoxoside have been shown to be cytotoxic against cancer cells at concentrations of up to 100 mg/mL. However, when hydrolysed to its aglucone, rooperol, by ß-3glucosidase, cytotoxicity was found to be ranging from concentrations between 2 and 10 mg/mL (Marini-Bettolo et al., 1982). This led to the discovery of the promising potential of Hypoxis hemerocallidea as an oral prodrug for cancer therapy in humans given its first-pass metabolism into non-toxic conjugate rooperol which may be activated in tumour cells with high deconjugase activity Albrecht et al. (1995b). Evidence of rooperol's activity as an anticancer agent has been further demonstrated using a model system consisting of normal, non-invasive breast epithelial cell lines by spontaneous immortalisation of cells from fibrocystic breast tissue support (Soule et al., 1990; Tait et al., 1990), and a premalignant, invasive and tumorogenic derivative, resulting in transfection of the cell line with a mutationally activated oncogene (Basolo et al., 1991; Ochieng et al., 1991). In a study by Boukes and Van de Venter (2011) to investigate and compare the cytotoxicity and mechanisms of action of three Hypoxis species, namely Hypoxis hemerocallidea, Hypoxis stellipilis and Hypoxis sobolifera var sobolifera, the authors concluded that different cell lines exhibit different sensitivities towards the plant extracts. They determined cytotoxicity of the three Hypoxis species against cervical (HeLa), colorectal (HT-29) and breast (MCF-7) cancer cell lines as well as peripheral blood mononuclear cells (PBMCs). They also determined DNA cell cycle arrest in the Gap 1 (G1), synthesis (S) or Gap 2/mitosis G2/M phase using propidium iodide staining. Hypoxis sobolifera was shown to be the most cytotoxic against all three cancer cell lines. The study provided the cytotoxic mechanism of Hypoxis, which they concluded is exerted, through the induction of cell cycle arrest and apoptosis (Boukes and Van de Venter, 2011). 2.1.5. Cardiovascular and anticonvulsant activity In a study involving Chacma baboons, Coetzee et al. (1996) established that a purified extract of Hypoxis hemerocallidea corms (rooperol) increased myocardial contractility in vivo. Rooperol caused moderate, transient increased cardiac output, stroke volume and vascular pressures without increased heart rate or filling pressures, suggestive of increased myocardial contractility probably allied to its catechol structure (Coetzee et al., 1996). Moreover, in their investigation on the effect of the aqueous corm extract of Hypoxis hemerocallidea on myocardial contractile performance of guinea-pig isolated atrial muscle strips in vitro, Ojewole et al. (2006) reported significant concentrationdependent positive inotropic and chronotropic responses. Furthermore, the extracts led to dose-related transient but significant (P o0.05–0.001) reductions in the systemic arterial blood pressure and heart rates of hypertensive rats used in the same study (Ojewole et al., 2006). In another study, findings report chronic ingestion of aqueous extract of Hypoxis hemerocallidea (as tea) to have caused ventricular tachycardia in a 25-year-old male subject (Ker, 2005). The observations suggest that Hypoxis hemerocallidea corm extracts contain some active chemical constituents with some cardiovascular activities. Ethanolic corm extracts of Hypoxis colchicifolia demonstrated dose-dependent inhibitory activity against the GABAA-benzodiazepine receptor complex involved in epilepsy and convulsions (Risa et al., 2004). Aqueous extracts of Hypoxis hemerocallidea (50–800 mg/kg) significantly delayed (Po 0.05–0.001) the onset of, and antagonised, pentylenetetrazole-induced seizures in mice better than phenobarbitone and diazepam, known anticonvulsant compounds (Ojewole, 2008). Stafford et al. (2007) reported ethyl acetate extracts of Hypoxis hemerocallidea to exhibit moderate non-selective inhibitory effects at IC50 of 25 75 mg/ml B. Ncube et al. / Journal of Ethnopharmacology 150 (2013) 818–827 on monoamine-oxidase enzyme which is involved in a number of neurological processes. 2.2. Phytochemistry and related bioactivities During the early 1970s, South African researchers realised the medicinal potential of Hypoxis and primed some chemical investigations. September 2013 marks 34 years since a detailed chemical analysis of Hypoxis hemerocallidea was conducted (Pegel, 1979; Drewes and Kahn, 2004). The most important and common phytochemical constituent in the Hypoxis species is a nor-lignan glycoside called hypoxoside [(E)-1,5-bis(4′-β-D-glucopyranosyloxy3′-hydroxyphenyl) pent-4-en-1-yne (CAS Reg no.: 83643-94-1)] (Albrecht et al., 1995b; Nair and Kanfer, 2007; Drewes et al., 2008) presented in Fig. 1. The history of the discovery of hypoxoside have been recorded in a review by Drewes and Kahn (2004). The review explains the circumstances leading to the first two publications on the constituents of Hypoxis hemerocallidea by Marini-Bettolo et al. (1982) working in Rome and Drewes et al. (1984) working in Pietermaritzburg, South Africa. Hypoxoside, when hydrolysed by the enzyme β-glucosidase readily converts to its aglycone, rooperol (Fig. 2), a biologically active compound that has therapeutic values (Drewes and Kahn, 2004; Mills et al., 2005). Hypoxoside has been reported to be largely localised in the corms, followed by lower levels in the roots and negligible concentrations in the leaves (Bayley and Van Staden, 1990). The compound was successfully tested in phase I trials as an oral pro-drug for cancer therapy and patented as an antitumoural and to treat viral infections (reducing the rate of decrease in CD4 lymphocyte counts), as well as an anaesthetic (Drewes and Kahn, 2004; Mills et al., 2005). In the Phase 1 trial, 19 out of 24 patients survived as long as their estimated prognosis (4 months), while 5 survived longer than expected (12 months to 5 years) (Smit et al., 1995). The patient who survived 5 years had no detectable metastases (Smit et al., 1995). It therefore seems possible that cancer patients who might benefit from hypoxoside are those with relatively slow-growing necrotising tumours that are inoperable and have high concentrations of f3-glucuronidase and sulphatase as well as a high sensitivity for rooperol, as was found for the H552 human adenocarcinoma cell line (Smit et al., 1995). Glycosides are the main constituents of Hypoxis species characterised by a common pent-1-en-4-yne backbone or a slight 823 modification of it. These include nyasol, nyasoside, nyaside, mononyasines (A and B), nyasicoside isolated from Hypoxis angustifolia and Hypoxis nyasica represented in Fig. 1 and Table 1. Other equally important compounds in the Hypoxidaceae family include obtusides and interjectin (Fig. 2), isolated from Hypoxis obtusa and Hypoxis interjecta respectively. The nor-lignan monoglucoside, nyasicoside was the first glucoside in which the glucose moiety was found linked to the aliphatic sequence instead of to the aromatic rings. Nyasol has been shown to possess anti-inflammatory activity in vitro and in vivo through the inhibition of cyclooxygenase (COX-2), inducible isoforms of nitric oxide synthase (iNOS) and 5-lipoxygenase (5-LOX). Nyasol is therefore, a broad spectrum inhibitor of eicosanoid and nitric oxide (NO) metabolism, without the capacity to down-regulate COX-2 and iNOS (Lim et al., 2009). Other reported biological activities of nyasol include inhibition of angiogenesis and hyaluronidase activity (Jeong et al., 1999, 2003), antioxidant and anti-artherogenic activities (Song et al., 2007), Fig. 2. Enzymatic hydrolysis of hypoxoside to rooperol. Fig. 1. Structures of some important glycosides isolated from Hypoxis species. 824 B. Ncube et al. / Journal of Ethnopharmacology 150 (2013) 818–827 Fig. 3. Structures of β-sitosterol, stigmasterol and stigmastanol isolated from Hypoxis species. anti-oomycete and antiviral activities (Park et al., 2003; Bae et al., 2007), antiallergic activity, such as LTB4 receptor antagonistic activity and inhibition of passive cutaneous anaphylaxis (Lee and Ryu, 1999; Bae et al., 2006). Nyasoside has been patented for the treatment of uterine cancer. Several other sterols including β-sitosterol, stigmasterol and their glycosides and stanols such as sitostanol (stigmastanol), presented in Fig. 3, have also been found in Hypoxis and have been reported to possess unconfirmed medicinal properties including anti-inflammatory, antipyretic, antioxidant, antiulcer, anti-cancer, anti-complement, antidiabetic and immune modulating effects (Bouic et al., 1996; Bouic and Lamprecht, 1999; Mills et al., 2005). As presented in Fig. 3, the structures of β-sitosterol, stigmasterol and stigmastanol are closely related thus giving them similar medicinal properties. Alone or in combinations, these sterols are used to treat increased serum total and low-density lipoprotein (LDL) cholesterol (Chol) concentrations, hypercholesterolaemia (Nieminen et al., 2008). β-Sitosterol inhibits cholesterol absorption in the intestine. Several steps are involved in the absorption of cholesterol by the intestine including solubilisation of unesterified cholesterol in mixed micelles composed of bile acids, phospholipids and products of triglyceride digestion (Christiansen et al., 2003). Mixed micelles are involved in carrying cholesterol through the water layer, which is the rate-limiting step in cholesterol absorption. When the sterol is absorbed in the intestine, it is transported by lipoproteins and incorporated into the cellular membrane. Because the structure of β-sitosterol is similar to that of cholesterol, β-sitosterol takes the place of dietary and biliary cholesterol in micelles produced in the intestinal lumen. Another mechanism involves the formation of an unabsorbable mixed crystal with cholesterol (cholestanol). The formation of cholestanol can however, facilitate the crystallisation of cholesterol leading to formation of gallstones or atherosclerotic plaques (Christiansen et al., 2003). 3. Safety and toxicity issues The toxicity of Hypoxis species have been investigated in several studies. Most of the studies confirmed lack of toxicity both after short and long-term use (5 years) of high doses of 2400 mg per day of plant extract (that is, 4 capsules 3 times a day) (Drewes and Kahn, 2004). Verschaeve et al. (2013), recently reported a lack of in vitro genotoxic effects of water extracts from four Hypoxis species (Hypoxis acuminata, Hypoxis colchicifolia, Hypoxis hemerocallidea and Hypoxis rigidula) and a commercial preparation thereof using the neutral red uptake assay, the alkaline comet assay and the cytome assay in human hepatoma HepG2 cells. They concluded that there was neither cytotoxicity nor genotoxicity caused by the extracts (Verschaeve et al., 2013). According to Drewes and Kahn (2004), the use of the species for a long time in traditional medicine on its own constitutes a form of ‘clinical trial’, since evidence of toxicity would have led to its abandonment by traditional healers long ago. In another study by Smit et al. (1995), a Phase I trial in cancer patients to investigate any clinical and biochemical toxicity that could be ascribed to the use of hypoxoside failed to produce any conclusive results. In yet another study, some clinical trials were prematurely terminated by the Data and Safety Monitoring Committee citing bone marrow suppression in patients (Mills et al., 2005). In vitro and in vivo studies involving guinea-pigs and rats on the aqueous extracts of Hypoxis hemerocallidea resulted in bradycardia and brief hypotension respectively (Ojewole, 2006). To add to the inconclusive reports available, further toxicity studies are important and urgently needed to clarify any molecular basis of isolated compounds, bioactive (transformed) compounds or crude extract toxicity if any. The bioactive constituent of Hypoxis hemerocallidea, rooperol derived from hypoxoside (as discussed above), is known to possess cytotoxic effects (Steenkamp and Gouws, 2006). In a study by Boukes and Van de Venter (2011) to investigate and compare the cytotoxicity and mechanisms of action of three Hypoxis species, namely Hypoxis hemerocallidea, Hypoxis stellipilis and Hypoxis sobolifera var sobolifera, the authors concluded that different cell lines exhibit different sensitivities towards the plant extracts. They determined cytotoxicity of the three Hypoxis species against cervical (HeLa), colorectal (HT-29) and breast (MCF-7) cancer cell lines as well as peripheral blood mononuclear cells (PBMCs). They also determined DNA cell cycle arrest in the Gap 1 (G1), synthesis (S) or Gap 2/mitosis G2/M phase using propidium iodide staining. Hypoxis sobolifera, was shown to be the most cytotoxic against all three cancer cell lines. The study provided the cytotoxic mechanism of Hypoxis, which they concluded is exerted through the induction of cell cycle arrest and apoptosis (Boukes and Van de Venter, 2011). Chronic infusion of Hypoxis hemerocallidea extracts was reported to result in a decrease in glomerular filtration rate, together with elevated plasma creatinine concentrations in rats, suggesting an impairment of kidney function (Musabayane et al., 2005). In addition, Hypoxis-induced immune suppression has been confirmed experimentally in the feline immunodeficiency virus model (Van Niekerk, 2003). A cardio-toxicity associated with cardio-active compounds in Hypoxis was reported in a patient with a known history of ischaemic heart disease who presented with ventricular tachycardia after ingestion of the aqueous extract (Ker, 2005). Caution should thus be exercised in the use of Hypoxis extracts for medicinal purpose at this stage. 4. Future prospects and potential pitfalls Judging from the extensive and diverse uses of the extracts and chemical constituents of Hypoxis for medicinal purposes, one gets to ask so many questions. What can plant species not be used for, given the general public beliefs and perceptions that they have to cure virtually any illness? If this is so, or becomes the case, what future do the species hold? Would they be able to survive the onslaught of modern living? What are the possibilities of species substitution? To the general public, the incessant media hype and the associated claims on the medical value of Hypoxis species, coupled with their long-time traditional use, further perpetuate the perceptions and stimulate their widespread use and trade. But, should B. Ncube et al. / Journal of Ethnopharmacology 150 (2013) 818–827 such public reactions be condemned and dismissed or is there medical value and benefits to it? To begin with, the documented history of medicinal usage of Hypoxis extracts can be traced from the 1960s (Drewes and Kahn, 2004) to date. Spanning from this period of more than half a century (53 years to be precise), perhaps a more logical approach to evaluate this outside the scientific paradigms is by assessing how many reported cases of poisoning are there against the treatment claims. Notwithstanding the fact that a sizeable number of poisoning/death cases as a result of using Hypoxis extracts might have gone unreported, their longtime use in traditional medicine to the present time is enough evidence to draw fundamental conclusions about the prospects it holds for the future. In this context, having considered the use of Hypoxis extracts, or the application of its pure components in the treatment of a number of diseases substantiated with numerous scientific and clinical trial evidence, it is conceivable to claim medicinal merits for the species. The fact that much of the positive pharmacological and clinical properties from several independent scientific evaluations have been reported compared to a few largely unsubstantiated claims of Hypoxis toxicity could serve as a platform to develop these extracts further. The major immune-system boosters, β-sitosterol and β-sitosterol glucoside, were originally obtained from Hypoxis extracts although, Bouic (1999), claims that in order to obtain sufficient of these active constituents from Hypoxis extracts would require the entire destruction of the species population to refine these molecules out of the total extract. How far from the truth could this be? A question that may possibly be best answered by extensive pharmacodynamic evaluations of Hypoxis extracts within the body system considering the fact that the crude extracts used in traditional medicine represent a hugely insignificant fraction of this suggestion. A case in point to reiterate this view is the remarkably stable CD lymphocyte counts concurrently with the decrease in serum p24 HIV antigen and expression of the HLA-DR CD8 lymphocyte activation marker on HIV patients administered with methanolic extracts from Hypoxis (Albrecht, 1996). The positive findings of this study lend pharmacological credence to the medicinal use of Hypoxis extracts against cancer and HIV. Additional to this, is the cardio-depressant and the transient hypotensive properties of the aqueous extracts of Hypoxis evaluated on guinea pig-isolated atrial muscle strips in vitro (Ojewole et al., 2007). Although the basic mechanisms of action of these extracts could possibly vary when applied in vivo through experimental animal models, the results points to existing potential of Hypoxis extracts as a natural remedy for management of cardiovascular ailments. This, among other positive scientific evaluation reports on the therapeutic values and potential of Hypoxis extracts holds a foreseeable future of the species in both traditional and modern medicine. Parallel to the positive pharmacological findings of Hypoxis extracts in the possible treatment of various ailments, Hutchings et al. (1996), relates to one of the traditional uses of Hypoxis colchicifolia corms as being ground and placed in food to kill small vermin. In principle or literally so, usage for such purposes implies presence of some toxic chemical constituents within these extracts yet they have been used and this continues for various other purposes. Could it be where part of the poisonous claims of the species emanates from? Moreover, Bouic (1999) reports that their research group terminated work on Hypoxis plants and cites among other reasons, the toxic effects of some extracts, and that extended use of the extracts could lead to bone marrow suppression in some individuals. In this regard, Verschaeve et al. (2013), reports a lack of in vitro genotoxic effects from water extracts of Hypoxis colchicifolia and three other Hypoxis species. However, should sound conclusions on the complete lack of toxicity of the species' extracts be drawn from studies that explore only a certain 825 aspect of toxicological evaluation? The answers is that extensive studies are still needed to draw valid conclusions with a wider applicability. In spite of the fact that several toxicity evaluations have been performed on several Hypoxis extracts and notwithstanding the importance that each of these contribute to the general understanding of the species' safety for medicinal use, a holistic approach to this aspect could perhaps provide the much needed answers. The toxic properties of an extract within a biological system are very complex and diverse and its complete elucidation requires an integration of different test procedures that encompasses different aspects of possible human poisoning effects. An incorporation of more pharmacokinetic and pharmacodynamic studies of every Hypoxis extract may offer a sustainable solution to possible pitfalls of ruling out the possibility of their toxic effects. As much as Hypoxis extracts and chemical constituents thereof may be used for medicinal purposes, the level of pharmacological activity, safety and toxicity will likely vary from one species to the next. What implications does this have given the fact that people use these as ‘Hypoxis extracts’ and in light of the morphological similarities among species? Species substitution is obviously one common phenomenon in such cases, unintentionally so in the case of species substitution due to misidentification and often deliberately so in cases where the demand for species is very high. An increased demand for the species for medicinal and commercial purposes leads to indiscriminate and unsustainable harvesting, with two possible negative consequences. The first of which, pertains to species substitution with either morphologically similar plant materials of the same genera or from different genera altogether. This is, in particular, so owing to the fact that most of the Hypoxis species used for medicinal purposes are collected from wild populations. The second negative aspect involves the ecological implications of unsustainable commercial harvesting on species survival and diversity in their natural habitats over time. Although several recommendations have to date been put forward towards the conservation of the species (Zschocke et al., 2000; Lewu et al., 2006; Ncube et al., 2011) implementation of practical conservation efforts remains to match the growing demand if Hypoxis species are to survive the onslaught of modern living. Commercial cultivation is one of the sustainable solutions that can address both issues, overexploitation of the species in the wild as well to safeguard against cases of poisoning through species substitution and to ensure quality. To achieve this goal, micropropagation protocols and in vitro optimisation procedures for secondary product biosynthesis have been established for some Hypoxis species (Appleton and Van Staden, 1995; Van Staden and Bayley, 1988; Appleton et al., 2012). The present situation on Hypoxis offers a lot of promising prospects for both traditional and modern medicine and is surrounded by possible potential pitfalls. Based on the evidence presented here, from a general public perception, it suffice to conclude and project that indeed Hypoxis is a potential herbal therapy for many ailments. Whether this extensive use of Hypoxis extracts translate to sound medical benefits remains an elusive fact to admit to in view of the diverse and complex nature with which these extracts are prepared and administered in the treatment of different ailments. Although scientific evaluations have yielded significant positive benefits of different extracts, herbal formulations and isolated pure compounds from Hypoxis, most of these studies employ mechanistic approaches and offer very little evidences to draw sound conclusions on the actual medical value of these extracts. Controlled clinical trials together with pharmacokinetic behavioural studies on different extracts, formulations and compounds in the treatment of different conditions would thus offer fundamental basis on their purported treatment claims. Quality control protocols to prevent misidentification and possible adulteration of Hypoxis species are an urgent necessity. 826 B. 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