Chapter 4 Downloaded by TSHWANE UNIV OF TECHNOLOGY on October 30, 2013 | http://pubs.acs.org Publication Date (Web): October 4, 2013 | doi: 10.1021/bk-2013-1127.ch004 Hypoxis hemerocallidea (African potato): A Botanical Whose Time Has Come? David R. Katerere* Department of Pharmaceutical Science, Faculty of Science, Tshwane University of Technology, Arcadia campus, Pretoria, 0001, South Africa *E-mail: david.katerere@mrc.ac.za. Hypoxis hemerocallidea is one of Southern Africa’s most important and popular medicinal plants. It is used for a wide a range of traditional medical treatments including urinary tract infections, heart disease, infertility and anxiety. It’s most popular contemporary use is for prostate disorders for which there is increasingly good evidence about it’s eficacy. Phytosterols are thought to be the main bioactive compounds for this indication and also for anti-lipidemic, anti-diabetic and anti-inlammatory properties exhibited by hypoxis extracts. Despite the popularity of hypoxis as a herbal and botanical medicine, the research into it’s pharmacological application has not been of suficient depth and width to prove it’s utility in modern medicine. Recent data using a colitis model presents an exciting new avenue which might inally yield products. However there continues to be a need for sustained in vitro and more importantly in vivo studies if this useful plant is be fully explored. Introduction Traditional medicine has experienced a resurgence in Africa in recent times. This is largely to do with a new sense of cultural pride following independence from colonial authorities who sought to suppress such practices during their tenure (1). In post-colonial Africa, there has also been limited access to allopathic medical services. Further, the emergence of new diseases such as HIV, and © 2013 American Chemical Society In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013. Downloaded by TSHWANE UNIV OF TECHNOLOGY on October 30, 2013 | http://pubs.acs.org Publication Date (Web): October 4, 2013 | doi: 10.1021/bk-2013-1127.ch004 previously unknown chronic diseases e.g. diabetes and cancer, most of which have no known medical cures has accelerated the growth of Complementary and Alternative Medicine (CAM) (2, 3) WHO estimates that 80% of the world’s population still largely relies on traditional medicine to meet it’s healthcare needs (4). South Africa has 7000 plants which are used in traditional medicine (5–7). One of the most famous, and certainly the most controversial is Hypoxis hemerocallidea. Commonly known as African potato, this herb was until recently widely promoted by a now deceased South African minister of health as an alternative to treatment of HIV / AIDS (8, 9) this despite little or no supporting scientiic evidence (10). Botanical Information Hypoxis hemerocallidea Fisch. & C.A. Mey, previously known as H. rooperi belongs to the family Hypoxidaceae which consists of 8 genera (11). Based on similarities with species of the Amaryllidaceae and Liliaceae family, the genera were previously classiied in these taxa (12). The hypoxis genus contains 130 species including H. hemerocallidea, H. acuminate, H. iridifolia and H. rigidula of which the irst is most widely used and is therefore the primary focus of this chapter. The moniker African potato is to some extent a misnomer as the rootstock is a corm rather than a tuber and the name causes confusion with tuberous Plectranthus esculentus (13). There is some evidence that the common hypoxis species are used interchangeably in traditional medical practice, sometimes inadvertently because of their close similarities (14). Hypoxis grows as a stemless, perennial geophyte with large black ibrous corms which are bright yellow inside when freshly cut (15). The leaves are organized typically into three ranks and maybe slightly hairy. The lowers which appear between October and January (16) are star-shaped and bright yellow and the number per inlorescence may vary from two to twelve (15, 17). While it is indigenous to southern Africa, hypoxis has a world-wide cosmopolitan distribution. It grows widely in the savanna grasslands of South Africa, Swaziland, Lesotho , Mozambique, Zimbabwe and into some parts of East Africa (11, 16). It has also been reported to grow in meanders, grasslands and mountains of South America, Australia and coastal areas of Asia (18). Traditional and Contemporary Use The rootstock of Hypoxis is the plant part of choice for medicinal and dietary use. It is the iconic African potato promoted by proponents of African traditional medicine in South Africa as a cure-all and derided by opponents as quackery (19) in the country’s highly charged political environment at the turn of the century. In Zulu traditional medicine it is widely known as “zifozonke” literally meaning “panacea” (20). It is known as African potato, starlower / South African star grass, Ilabatheka, Inkofe (in Zulu), sterretjie (in Afrikaans), moli kharatsa, monna wa maledu, thitidi (in Sotho), hodzori (in Shona) (21). 52 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013. Downloaded by TSHWANE UNIV OF TECHNOLOGY on October 30, 2013 | http://pubs.acs.org Publication Date (Web): October 4, 2013 | doi: 10.1021/bk-2013-1127.ch004 The aqueous infusion is used as tonic in children and to treat dizziness and mental disorders in adults (16). Hypoxis is widely used traditionally particularly in Zulu society for heart disease, bad dreams, anxiety, insanity, barrenness, intestinal parasites, urinary tract infections among other indications (11, 22). Related species mainly H. colchicifolia (syn. H. oligotricha), H obtuse, H. nyasica and H. angustifolia are also used for similar indications (11). In addition H. obtuse, which more commonly grows north of South Africa’s border is used traditionally for infertility in women, abdominal pains, heart pains, bile emesis, gonorrhea and for aphrodisiac effects (21). Hypoxis also has ethnoveterinary uses. In South Africa Hypoxis hemerocallidea and H.rigidula have been reportedly used for fertility enhancement, general ailments, heartwater, and to prevent abortion in cattle by the Tswana people (23) . Kambizi (24) has reported that fresh cut corms are applied to the face by Xhosa women as facial treatments or used to treat burns. In contemporary society, the use of hypoxis encompasses many of the traditional uses and more recent indications. Today it is marketed both in its crude unprocessed form (at traditional medicine (so-called muthi) markets) (7) and also in pharmaceutical formulations and packaging as a phytomedicine in retail pharmacies. In both formats the marketing materials carry many unsubstantiated claims viz. HIV / AIDS, arthritis, myalgic encephalomyelitis (ME), hypertension, asthma, diabetes mellitus, cancer, arthritis, psoriasis, tuberculosis and epilepsy (22, 25–28). It is also increasingly being formulated into dermatological products with purported antiviral and antifungal actions (11). In Germany, hypoxis extracts have been widely marketed since the late 1970’s for the treatment of prostate adenoma and benign prostate hypertrophy (BPH) (11, 29, 30). This indication is based on the inhibition of 5α-reductase metabolism of testosterone by phytosterols from hypoxis (11). Dold and Cocks (7) estimated that trade in hypoxis exceeds 11 metric tons per year in South Africa alone. Because of the high demand, hypoxis populations are declining and under pressure (31). The use of corms is particularly destructive and (32) investigated the possible use of the aerial parts as substitutes. They found clear differences between the two plant parts both in chemical constitution and biological activity making the substitution of corms for leaves less attractive. Phytochemistry Several compounds have been isolated from hypoxis species and some of them have been linked to their putative biological activity. Hypoxoside, a norlignan diglucoside is an aglycone consisting of a diphenyl-1-en-4-yne-pentane unit (33) and has been isolated from all the common Hypoxis species (34). have suggested that this compound maybe unique to the genus. Hypoxoside is hydrolysed by β-glucosidase to rooperol which appears to be the active bioavailable compound (26). However it is for its high concentration of phytosterols that hypoxis is best known for, especially sterols, sterolins and stanols. The patent by (29) claimed that aqueous extracts of H. hemerollicadea had phytosterol yield of 9 mg / 53 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013. Downloaded by TSHWANE UNIV OF TECHNOLOGY on October 30, 2013 | http://pubs.acs.org Publication Date (Web): October 4, 2013 | doi: 10.1021/bk-2013-1127.ch004 100g, however this could not be reproduced in recent studies by (13). These workers found less than 0.01% w/w inal extract recovery they have dismissed the therapeutic importance of sterolins in hypoxis based on that data. The likelihood is that they were not using the same material and hence the differences are inconclusive. The plant hormone (cytokinin) zeatin and it’s derivatives were isolated from the corms by Van Staden (35). Biological Activity It is fair to say that because of its high proile status in South African materia medica, hypoxis has been well studied for its biological effects (Table 1). Methanol extracts of H. hemerocallidea corms showed no activity against Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis (14). Using freshly harvested corms grown in the laboratory (32) previously showed that acetone and ethanol extracts of the leaves had relatively good activity against both E. coli (MIC < 0.31 mg /ml) and to some extent Pseudomonas aeruginosa (MIC < 0.61 mg / ml). The aerial parts of hypoxis have not been previously investigated so this study was important in showing their possible utility. Steenkamp et al. (36) showed inhibition of E. coli by both water and ethanolic extracts of H. hemerocallidea with MIC of 62.5 ug / ml, though they estimated that it would not translate to a therapeutic dose in humans (based on the dosage). They further suggested that the antimicrobial activity maybe another reason why hypoxis is used in prostatitis and prostate indications. Hypoxoside and rooperol showed good activity against E. coli and S. aureus, with the latter being more active than the positive control (neomycin) (37). They also possess good antimuagenic and cytotoxic properties (38) Sathegke (14) showed that methanol extracts of hypoxis is non-toxic in a cytcoxicity assay done using Vero cells (African green monkey kidney cell line) and no activity was seen in HeLa cancer cell line. H. iridifolia and H. rigidula showed slightly better activity in the HeLa cytotoxicity assays. Albrecht et al (26) have previously reported that rooperol, the hydrolytic product of hypoxoside showed activity against HeLa cells under 10 µg/ml, while the glycoside was inactive in the same cell line. Cytotoxic activity against HeLa, HT-29 and MCF-7 cancer cells appeared to increase when β-glucosidase was added to H. hemerocallidea which seemed to imply that there were certain compounds (other than hypoxoside) which were being hydrolyzed to active metabolites (39) Van der Merwe et al (40) demonstrated that rooperol inhibits leucotriene synthesis in leukocytes and prostaglandin synthesis in platelet microsomes, thus showing in vitro antioxidant and anti-inlammatory activity. Good free radical scavenging ability was of aqueous and ethanolic extracts of H. hemerocallidea was shown by Steenkamp et al (36). Antioxidant and free radical scavenging activities of rooperol has been conirmed in various assays (11, 18, 33) as would be expected of most phenolic compounds. 54 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013. Downloaded by TSHWANE UNIV OF TECHNOLOGY on October 30, 2013 | http://pubs.acs.org Publication Date (Web): October 4, 2013 | doi: 10.1021/bk-2013-1127.ch004 Ojewole (25) found that an aqueous decoction of the corms of hypoxis had signiicant antinociceptive and anti-inlammatory activity which was dose dependent (50 – 800 mg / kg p.o.). In the same study, hypoxis extract showed hypoglycemia in both normal and streptozotocin (STZ)-induced diabetes mellitus rats. Gaidamashvili and Van Staden (41) showed that lectins isolated from H. hemerocallidea may partly explain the anti-inlammatory activity as it elicited a 29% inhibition of cyclooxygenase enzymes which catalyze the conversion of arachidonic acid to prostaglandin. In a study by Laporta et al (37), using concentrations below 0.5 mg / ml, there was no inhibition of COX-1 and COX-2 by either the crude extract or the compound hypoxoside. Rooperol, however showed strong inhibition against both COX enzymes with a COX-2 / COX-1 IC50 ratio of 2. It is thought that the compound exerts it’s anti-inlammatory actions by also inhibiting the production of NF-κB, AP-1 and similar transcriptional factors important in inlammatory processes (42) Ojewole (43) has attempted to validate the ethnobotanical use of hypoxis in managing hysterical its and epilepsy. Using a rat model, he found that aqueous corm extracts (100 – 800 mg / kg i.p.) delayed the onset of, and signiicantly inhibited, seizures. In other similar studies in rodent models, Ojewole and his colleagues tried to validate the use of hypoxis in gastroinstestinal and uterine – related conditions, speciically, how do aqueous extracts of hypoxis modulate smooth muscle? (44–46). They found that hypoxis extracts reduced gastrointestinal motility, the frequency of defaecation and hence the severity of diarrhea in experimental rats (43). The uterolytic effects were also conirmed (46). In these experiments the muscle relaxant effects were found to be refractory to β-adrenoceptor, cholinergic and histaminergic blockers leading the workers to conclude that the mode of action might be through a non-speciic spasmolytic mechanism. The dichloromethane (DCM) extract of H. colchicifolia leaves was the only one with signiicant activity of 16 plant extracts assayed for anthelmintic activity using an in vitro colorimetric assay measuring free-living nematode larvae viability (47). H. hemerocallidea was inactive in this study. A systematic review by (48) of various phytotherapies used to ameliorate the symptoms of Benign Prostatic Hyperplasia (BPH) Serenoa repens (saw palmetto), Pygeum africanum (African plum) and H. hemerocallidea, showed that the latter two improved urinary low rates and symptoms compared to placebo. Several clinical studies have demonstrated that the use of hypoxis extracts in BPH patients resulted in signiicant sustained symptomatic relief (49). The effects of hypoxis on the prostate have always been linked to the purported occurrence of high concentrations of phytosterols. These compounds are known to inhibit 5α-reductase, the enzyme catalyzing the conversion of testosterone to dihydroxytestorone (DHT). BPH is prevalent in 50% of men over 50 years of age and in 90% of men over 80 years old and it is known to depend on androgens, especially DHT (50). Inhibitors of 5α-reductase can also be used for male pattern baldness, hirsutism and prostate cancer. Hypoxis has been widely marketed and used for prostate disorders but there is need for more studies especially in light of recent indings that concentration of phytosterols in the species may actually be much lower than 55 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013. Downloaded by TSHWANE UNIV OF TECHNOLOGY on October 30, 2013 | http://pubs.acs.org Publication Date (Web): October 4, 2013 | doi: 10.1021/bk-2013-1127.ch004 previously recorded (13). When this is considered in the light of the eficacy demonstrated in the systematic review of Dedhia and McVary (48), it may imply that other compounds which are yet to be identiied or tested are responsible for the purported activity. Phytosterols from hypoxis (mainly stigmasterol, β-sitosterol, brassicasterol and ergosterol) are also marketed for their anti-lipidemic, cholesterol - lowering actions. They accomplish this inhibiting Delta24 ( δ 24) reductase enzymes in the cholesterol synthetic pathway (51). The lipid lowering effects of phytosterols are however equivocal because the bioavailability of these compounds is variable and dependant on formulation and other co-ingested foods (39) The putative anti-inlammatory, antidiabetic and immune modulating properties of hypoxis have been attributed to β-sitosterol (52). The amount of sitosterol in hypoxis on average is variable. A study by (53) on immune modulation showed that hypoxis extract was inferior to control in the survival rates of FIV+ (Feline immunodeiciency virus infected) laboratory cat model. However, a sitosterol enriched product showed signiicantly improved survival rates. Toxicology and Adverse Effects The African Herbal Pharmacopeia (54) suggests that oral ingestion of hypoxoside showed no toxicity, and no lethality was seen in the brine shrimp assays nor cytotoxicity in Vero monkey cells. However, it would appear that the use of crude herbal extracts of hypoxis has caused some concern. A study on renal function of wistar rats after both acute and chronic exposure to aqueous H. hemerocallidea extracts found that there was impaired function with signiicant decrease in glomerular iltration rate (GFR) and increased plasma creatinine concentration (55). A human study on the safety and eficacy of hypoxis extracts in HIV positive patients in South Africa was prematurely terminated by regulatory authorities reportedly because of bone marrow suppression and immune suppression which has been also been reported in a feline study (56). Steroids are known to be both immunesuppressive and may cause osteoporosis (57) and this maybe evidence of the high concentration of steroidal compounds in hypoxis. In mice the LD50 of aqueous extracts of the corm of hypoxis was about 2g / kg meaning that the species shows low acute toxicity (58). Low doses of alcoholic and aqueous extracts of hypoxis, while causing a signiicant increase of weight in suckling rats, did not show any morphometric changes in the small intestine of the test animals (59). These workers concluded that hypoxis had no adverse effects on suckling rats. Dichloromethane and 90% methanol aqueous extracts of hypoxis showed no mutagenic or antimutagenic activity in the Ames Salmonella / microsome mutagenicity assay (60). Daily doses of up to 3.2 g of hypoxis extract showed no clinical toxicity or adverse changes in biochemical or haematological measurements in a phase I clinical trial in human lung cancer patients (61). While hypoxis has generally been 56 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013. Downloaded by TSHWANE UNIV OF TECHNOLOGY on October 30, 2013 | http://pubs.acs.org Publication Date (Web): October 4, 2013 | doi: 10.1021/bk-2013-1127.ch004 proved to cause little toxicity, it is the potential for drug-herb interactions which still requires more extensive research. This is an increasingly important direction of research as the concomitant ingestion of herbs with pharmaceuticals is common especially in the treatment of HIV / AIDS (62). Therefore the potential for drugherb adverse reactions is high. Nair et al. (10) have shown that hypoxis inhibits human drug metabolizing enzymes, CYP3A5, 3A4 and CYP19 by 11%, 33% and 41% respectively while rooperol showed almost total inhibition of CYP3A4 and 3A5. At concentrations of 5 mg / ml, hypoxis extracts showed high induction of the polyglycoprotein (Pg-p) transporter. No effect was shown on Pg-p by sterols or rooperol (10). Future Directions of Research Liu et al (42) have recently for the irst time used a colitis model to demonstrate that methanolic extracts of hypoxis ameliorates severe typhlocolitis in mice. The extract reduced weight loss, severity of bacteria – induced colitis, neutrophil iniltration and intestinal epithelial proliferation making hypoxis and it’s constituents an interesting lead in the search for prophylactic therapies for inlammatory bowel diseases. However the extract did not show similar eficacy in a non-bacteria-induced, dextran sodium sulfate (DSS) – induced colitis model. This is certainly an interesting and innovative direction in which research can move. There remain many unanswered questions e.g. environmental variation and how it impacts on the chemistry and biological activity of hypoxis, the apparently contentious issue of the amounts of sterols and sterolins and their putative activity. Despite all the work that has been done so far, there remains a need to understand the phytochemistry of hypoxis in it’s entirety and then to test the compounds in various in vitro and in vitro assays. Finding the bioactive compound (s) may lead to the progression of such a compound in the pharmaceutical drug pipeline and /or to the standardization of hypoxis extracts. Conclusion Hypoxis hemerocallidea is an important medicinal and dietary plant in Southern Africa. However it’s contemporary use (particularly in HIV / AIDS and so-called immune-boosting products) has been controversial and political. This may have compromised the scientiic research into it’s use in these areas. There are some important data which have been gathered from in vitro and rodent studies which may form a good basis for the design of further pre-clinical studies. However there is a general lack of extensive phytochemical studies which may be the irst step towards developing evidence – based botanical / phytomedicine products. 57 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013. Acknowledgments I wish to thank Dr. Learnmore Kambizi (CPUT) for proofreading the inal draft and useful comments. References Downloaded by TSHWANE UNIV OF TECHNOLOGY on October 30, 2013 | http://pubs.acs.org Publication Date (Web): October 4, 2013 | doi: 10.1021/bk-2013-1127.ch004 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Chavunduka, G. Traditional Medicine in Modern Zimbabwe; University of Zimbabwe Publications: Zimbabwe,1994. 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