Extracts of Hypoxis hemerocallidea corm (African potato) are commonly used by some traditional health practitioners in KwaZulu-Natal Province of South Africa for an array of human ailments. This study was, therefore, undertaken to investigate the GIT spasmolytic, bronchospasmolytic, uterolytic and vasa deferentia relaxant effects of Hypoxis hemerocallidea corm aqueous extract. Respectively, these effects were determined on both naive and spasmogenevoked contractions of the guinea-pig and rat isolated ileum, trachea, uterine horns and the vas deferens in vitro. Healthy, young adult, male and female Dunkin-Hartley guinea-pigs (300-400g) and Wistar rats (250-350g) were used in this study. The isolated tissues were prepared and mounted in Ugo Basile organ-baths under normal physiological conditions. After an equilibration period of 30-45 minutes, the isolated smooth tissue segments were challenged with graded concentrations of Hypoxis hemerocallidea corm aqueous extract, and/or reference drugs. Changes in tension developed by the muscle preparations (relaxations and contractions) were recorded isometrically by means of Ugo Basile's force-displacement transducers and pen-writing 'Gemini' recorders. Relatively low to high concentrations of Hypoxis hemerocallidea corm aqueous extract (APE, 25-400 mg/ml) produced dose-dependent and significant (p<0.05) relaxations of the guinea-pig ileum, and the uterine horns taken from non-pregnant rats, as well as on spasmogenprovoked contractions of stilboesterol-primed, oestrogen-dominated, non-pregnant rats in a concentration-related manner. Potassium chloride (40 mM)-induced contractions of uterine horns, ACh (0.1-3.2 ug/ml)-induced increases in the amplitude of contractions of the guinea-pig ileum, as well as noradrenaline (0.2-1.6 ug/ml)-induced increases in the amplitude of contractions of the male rat isolated vasa diferentia, were significantly (p<0.05-0.001) reduced or abolished by bathapplied APE (25-400 mg/ml). Relatively low to high concentrations of the extract (25-400 mg/ml) caused concentration-dependent increases in the relaxations of the guinea-pig isolated tracheal smooth muscles. Inhibitions of ACh (0.1-3.2 ug/ml)-induced contractions of the guineapig isolated ileum probably suggests possession of antidiarrhoeal activity of APE. Results of this study show pronounced relaxant effects of Hypoxis hemerocallidea corm aqueous extract on guinea-pig vas deferens. The study also lends pharmacological credence to the folkloric, ethnomedical uses of APE as a natural antenatal remedy for threatening abortions, as an antidiarrhoeal remedy, and as a bronchorelaxant. The precise mechanisms of APE action on the smooth muscles could not be established in the present study. However, the uterolytic action of the corm's extract is unlikely to be mediated via ^-adrenoceptor stimulation, but probably mediated through a non-specific spasmolytic mechanism. / Thesis (M.Pharm.)-University of KwaZulu-Natal, 2009.

Thesis (MTech (Horticulture))--Cape Peninsula University of Technology, 2017. / The exact responses to the concentration of compost tea extract and methods of irrigation application were not previously measured on Hypoxis hemerocallidea and Siphonochilus aethiopicus. Commercial exploitation, habitat loss and degradation, overharvesting, and enhancement of their medicinal properties, have led to this investigation and the need to replenish both these valuable plant species. This is crucial for plant survival, especially in the wild and for use of the traditional medicinal plants. Hypoxis hemerocallidea and Siphonochilus aethiopicus, known as star flower and wild ginger respectively continue to decline, due to overharvesting from their natural habitat. Both these species have tremendous traditional medicinal value among localized African people. To enhance their commercial cultivation, compost tea extracts, in the following ratios (no catalyst added (control1); T 1000:1, T750:1, T500:1, and T250:1L) were applied in equal dosages to determine an optimal compost tea extract ratio. The experiment was conducted in a temperature controlled greenhouse. Mushroom compost (500 g, per brew) was used for all extracts. Brewing was done with no catalyst added (Control 1), and 24 hours later another brew was done with catalyst added, weekly for 20 weeks. The Control treatment received water only. Both species were slow growing and comparatively, the Hypoxis plants responded faster than the Siphonochilus plants. In this investigation, plant growth parameters such as plant height, number of leaves, leaf width, leaf length and leaf colour, were measured and evaluated. Despite the plants positive response to the mushroom compost tea extracts, across all the above plant growth parameters, no significant differences were noted between the treatments during the twenty-week application period. Leaf chlorophyll content peaked in week 11 of the hypoxis plants and was the highest in week 14 of the Siphonochilus plants with no significant interaction between weeks vs. treatment over the twenty-week experiment. The chlorophyll readings indicate that both species increased their chlorophyll production over time. Although the total wet leaf length, root length, corm diameter, leaf weight, corm weight and root weight of Hypoxis were non-significant between treatments, T500:1 total wet weight was significantly higher when compared to the rest of the treatments. The total dry weight analysis of hypoxis was non-significant. Control Calcium level was significantly lower between the control and the treatments of the Hypoxis total dry leaf nutrient analysis. The Hypoxis dried roots nutrient analysis was non-significant across treatments.

Hypoxoside, a phenolic diglucoside, with a diarylpentane-type structure, is thought to be the medicinally active constituent of corm extracts of Hypoxis hemerocallidea Fisch. & Mey. which are reputed to alleviate the symptoms of prostate hypertrophy and urinary infections. The biosynthes is and production of this unique phytochemical were investigated in H. hemerocallidea using both in vivo and in vitro systems. It was found, in root-producing callus, that [l4]C-phenylalanine and [14]C-t-cinnamic acid were efficient precursors for hypoxoside in comparison to [14]C-sodium acetate and [14]C-acetyl coenzyme-A, which were not incorporated into the phenolic compound. Thus, at least one aryl moiety of hypoxoside was derived, via phenylalanine and t-cinnamic acid, from the shikimate pathway. The acetate pathway did not appear to be involved in the biosynthetic process. The data supports the hypothesis that the molecule is formed from two cinnamate units with the loss of a carbon atom, in opposition to the proposal that the molecule is derived from head-to-tail condensation of acetate units onto a propenylic moiety. Despite the structural similarities between hypoxoside and caffeic and p-coumaric acids, these two hydroxycinnamic acids were not efficient precursors for hypoxoside in vivo or in vitro. A number of reasons are put forward to explain this finding. It was found that the greatest concentration of hypoxoside was located in the corms of intact plants. The major biosynthetic site of the molecule was also found to be located in this organ. Since the roots did accumulate the phytochemical to a small extent, the biosynthetic potential of these organs has not been disregarded. That of the leaves has been, however. The report by PAGE (1984) that the upper region of the corm contained a greater con cent ration of hypoxoside than the lower portion, is substantiated in this study, where this region was found to be more biosynthetically active than the lower half. Light microscopic and electron microscopic studies revealed that starch storing cells, which accumulated phenolics in their vacuoles, contained seemingly synthetically active tubular endoplasmic reticulum in their cytoplasm. A greater number of these cells were concentrated in the upper region as opposed to the lower half of the corm. It is postulated that these cells are the site for biosynthesis and accumulation of hypoxoside. The shikimate pathway, from which the precursors for hypoxoside are derived, was found, through the exposure of intact plants to [14]C-carbon dioxide, to be located mainly in the leaves. It is postulated from the above study and one in which [14]C-phenylalanine, [14]C-t-cinnamic acid, [14]C-p-coumaric acid and [14]C-caffeic acid were applied to intact plants, that phenylalanine and/or cinnamic acid are the transported form of the shi kimate derivatives. p-Coumaric and caffeic acids, which are metabolically more stable, are envisaged to be the sequestering forms. The investigation of the seasonal production of hypoxoside revealed that most of the synthesis and accumulation occurred after the corms had broken winter dormancy and after the flush of leaf growth had slowed down. During dormancy the production of hypoxoside appeared to cease. The in vjtro studies, where the effects of light, temperature, nutrients, plant growth regulators and supply of potential precursors, on hypoxoside production by root-producing callus were investigated, indicate that this metabolite is not simply a "shunt" metabolite. A number of factors other than precursor availability enhanced, or reduced the jn vjtro production of this phytochemical. Furthermore, production of the phytochemical and growth were not always antagonistic. Hypoxoside, the biosynthesis of which requires a more thorough investigation, is, however, according to this investigation, a typical secondary metabolite in many respects. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1989.

The large geophytic monocotyledon, Hypoxis colchicifolia Baker, has been identified for the importance of its corm extracts in the development of a potential non-toxic prodrug for the treatment of inflammation, certain malignancies and HIV-infection. The underground corms of this plant are also commonly used for therapeutic applications in traditional medicine in Kwazulu-Natal where it primarily occurs. A review of published literature revealed, however, that H. colchicifolia plants are currently harvested in an unsustainable manner from traditional collecting sites due largely to population growth, increased land use for urban development and agriculture, and the popularisation of Hypoxis plants for herbal remedies. A further search of historical records established that H. colchicifolia plants were dominant in grassland vegetation prior to 1950, but had rapidly declined since then. Quantitative data subsequently gathered in this study from comparative surveys of both H. colchicifolia and H. hemerocallidea populations from sites with near-pristine, disturbed, burnt and mown grassland vegetation showed for the first time that exposure to human activity and the grassland management practices of mowing and burning incurred not only a 75% reduction in plant density of both these Hypoxis species, but also the total destruction of mature plants of H. colchicifolia in frequently mown and burnt areas. Flowering data recorded in these surveys, and confirmed by monitoring field performance of cultivated H. colchicifolia plants, showed that a contributing factor to the plant's inability to withstand these pressures was that juvenile forms only reached flowering maturity after three to four years growth, thus adversely affecting seedling recruitment. It was concluded therefore that, since Hypoxis species responded differently to mowing and burning, geophytic plants should be considered individually and not as "forbs" during the planning of grassland management programmes for natural conservation areas. The need to cultivate H. colchicifolia to ensure its survival was also established using the new field data gathered in this study. Methods to propagate this species have, however, not been established. Data gathered on all the plants comprising a single population confirmed that mature plants survive to an estimated 20 years and longer in natural areas. Greatest hypoxoside yields were also obtained from corms with a fresh mass of 350g to 400g. Since these corms were estimated to be 10-years-old and older, propagation and cultivation methods that could sustain plant production and survival for long periods, and therefore increased hypoxoside yields, would have to be developed. Several micropropagation systems suitable for the mass production of H. colchicifolia and from which phenotypically normal plantlets were recovered, were therefore established via organogenesis, embryo culture and somatic embryogenesis. The latter cultures have not been reported previously for Hypoxis. In the former culture the toxic effects of phenolic leachates and browning were controlled, and improved plantlet regeneration achieved, by adding polyvinyl pyrrolidone to the medium and introducing distinct sequential aseptic steps into the micropropagation procedure developed. Defined protocols for the different phases of in vitro somatic embryogenesis are not readily available for monocotyledons, however, neither are the factors controlling embryogenesis and organ regeneration known. In this study the process of somatic embryogenesis from excised zygotic embryos of H. colchicifolia was shown to be complex and the resultant cultures very heterogeneous. Although the stage of development of the zygotic embryo explants was important at the time of inoculation, data showed that the induction and regulation of the processes of embryo culture and somatic embryogenesis were ultimately determined by the exogenously applied plant growth regulators. By comparing the different pathways leading to plantlet regeneration, and the morphological stages of development of the structures produced both on solid and in liquid media, not only photographically, but also quantitatively and schematically, the repeated formation of pseudoembryonic structures and neomorphs confirmed that they form an integral part in the in vitro somatic embryogenic pathway of H. colchicifolia. Evidence suggested not only that two types of somatic embryos are produced in the embryogenic cultures of H. colchicifolia, but that the pseudoembryonic structures produced resemble the pseudobulbils produced in polyembryonic cultures of Citrus. The success of the somatic embryogenic cultures was confirmed by the estimation that 28 112 somatic embryos and embryo clusters of H. colchicifolia could be obtained from 16 ml of somatic embryogenic liquid culture. Furthermore phenotypically normal plantlets regenerated from all of the micropropagation procedures developed were successfully transplanted from the laboratory, acclimatized under greenhouse conditions and their horticultural and field performances evaluated. / Thesis (Ph.D.)-University of KwaZulu- Natal, Pietermaritzburg, 2004.

>Magister Scientiae - MSc / About 180 million people have been estimated to suffer from type 2 diabetes (T2DM) in 2006 and the annual death rate due to this disease was 3 million by that time. More than 400 medicinal plants used for the treatment of diabetes mellitus have been recorded, but only a small number of these plants have received scientific and medical evaluation to assess their efficacy. The most common plant used to treat diabetes mellitus is Hypoxis hemerocallidea (HH). The present study was undertaken to investigate the effects of Hypoxis hemerocallidea (HH) on T2DM in rats. Male Wistar rats weighing 200-250 g were used in this experiment. Hypoxis hemerocallidea (HH) corm was used as plant material in the experiment. The study was based on three parts, an acute diabetes study, chronic diabetes study and insulin secretion study. In the acute study, the rats were randomly divided into 2 groups (control and diabetes). The saline solution was added to different concentrations of HH corm to produce concentration of (50, 200, 400, 800 mg/ml). Diabetes was induced by intraperitoneal injections of STZ (65mg/kg). Two weeks after the injection (STZ 65 mg/kg), different concentrations of HHS was administered intraperitoneally after an overnight fast. The blood glucose levels were monitored in the diabetic and control rats at, 30, 60, 120, 180 and 240 minutes post injection. In the chronic study, the rats were randomly divided into 6 different groups (control, HFD, DM, DM-HH, DM-PTHH, and HH). Diabetes mellitus was then induced in the groups of diabetic rats by intraperitoneal injections of STZ (40 mg/kg) and rats were fed a high fat diet (HFD). The body weight of the rats were measured weekly for 7 weeks. An intraperitoneal glucose tolerance test (IPGTT) was performed at the end of week 6. At the end of week 7, rats were killed and serum sample were collected for determination of fatty acid and insulin. Liver and pancreatic tissue was collected for histological evaluation. In the insulin secretion study, Hypoxis hemerocallidea was tested for its effects on insulin secretion by pancreatic islet cells exposed to low (3mM) and high (20mM) glucose medium. Results of the acute study indicated that HHS at a dose 800 mg/ml decreased blood glucose levels fastest in both normal and diabetic rats reaching significance after 30 minutes and 60 minutes respectively and remained below the baseline value until 240 minutes. In the chronic study, it was illustrated that HH had no effect in normal rats on any of the parameters evaluated. Animals in the DM group gained weight the first two weeks, but thereafter began to lose weight. At the end of seven weeks the animals gained significantly less weight than the rest. Animals fed a HFD have more visceral fat compared to the control group. The visceral fat gain occurred in the absence of a significant increase in body weight. We found a markedly lower fasting glucose level in HH treated diabetic animals compared to untreated DM animals. At time zero the blood glucose level of the HFD group (5.8±0.5mmol/l) and the HH group (4.9±0.7mmol/l) were in the normal range, and were not significantly different (P > 0.05) from the control group (5.0±0.2mmol/l). After glucose load peak blood glucose levels was measured after 30 minutes in the control group (9.0±0.6mmol/l), the HFD group (9.8±0.4 mmol/l), the DM-HH group (21±5.7 mmol/l) and the DM-HHPT group (27.8±5.3 mmol/l). In the HH group the blood glucose level reached a peak at 60 minutes (7.6±0.6 mmol/l). In the DM group two peaks were recorded one after 10 minutes (27.2±7.1mmol/l) and another after 60 minutes (31±5.2 mmol/l). In the groups control, HFD, DMHH, DM-HHPT and HH groups the blood glucose level after 120 minutes were not significantly different from the time zero value. The blood glucose level after 120 minutes in the DM group (28.2±7.1 mmol/L) was significantly higher (P ≤ 0.01) than from the time zero value. Serum fatty acid levels were increased in all groups fed a high fat diet. The serum insulin levels in the HFD group (6.2 ± 0.76 μUI/ml protein; P ≤ 0.05 ), the DM group (2.0 ± 0.9 μUI/ml protein; P ≤ 0.001), the DMHH group (3.4 ± 0.7 μUI/ml protein; P ≤ 0.001) and the DM-HHPT group (3.0 ± 1.1 μUI/ml protein; P ≤ 0.001) were significantly lower than the control group. The β-cell function in the HFD group (62 ± 8 %; P ≤ 0.001), the DM group (3 ± 1 %; P ≤ 0.001), the DM-HH (11 ± 9 %; P ≤ 0.001) group and the DM-HHPT group (4 ± 2 %; P ≤ 0.001) were significantly lower than the control group. The histological observation of the liver and the pancreas in rats after 7 weeks on different dietary regimes showed some morphological changes within the liver and pancreas parenchyma of some rats. In the insulin secretion study, glucose stimulated insulin secretion in low (3mM) and high (2mM) glucose concentration. Furthermore, insulin secretion was significantly higher when the glucose concentration was increased from 3mM to 20 mM (1.10 ± 0.13 μUI/ml protein and 1.5 ± 0.17 mIU/mg protein respectively P≤ 0.01). In the presence of low HH (100 µg/ml), there was a marked increase in insulin secretion when exposure to high glucose compared to low glucose concentration, while in the presence of high HH (500 µg/ml), there was no significant different in insulin secretion in the presence of low or high glucose. In conclusion, the results of this experimental study indicate that a concentration 800 mg/kg of HHS produces maximal hypoglycaemic effect in fasted normal and diabetic rats. HH has an antidiabetic activity as it lowers serum glucose levels in T2DM rats and significantly increases glucose tolerance. It also increases body weight of diabetic rats. HH treatment was found to improve insulin secretion in pancreatic islet cells.

Phytosterols and their glucosides (sterolins) have many therapeutic indications e.g. immune modulation, hypercholesterolaemia and benign prostatic hyperplasia (BPH). In this study sterolslsterolins in three BPH phytotherapeutics (Hypoxis hemerocallidea, Prunus africana and Serenoa repens) and related products were investigated. The aim of this study was to develop, evaluate and apply TLC and HPLC methods for the qualitative and quantitative analyses of sterols and sterolins. A new optimum TLC method was developed for good visibility and separation of phytosterols and sterolins and could be used to qualitatively compare sterol/sterolin content. A published HPLC method to determine the bioavailability of β-sitosterol in humans was used in a new application to quantitatively determine phytosterols in plant extracts. A new and sensitive method to determine hypoxoside (norlignan diglucoside unique to Hypoxidaceae), by isolation from the crude methanol extract with solid phase extraction (SPE) and HPLC quantification using fluorescence detection (excitation wavelength of 230 nm and emission wavelength of 345 nm), was developed. The developed TLC and adapted HPLC methods were applied to determine the stability of phytosterols, subjected to increased temperature and gamma irradiation. Phytosterols in isolated form were more stable than the phytosterols in plant material. The data from the accelerated stability tests could be used to estimate the shelf-lives of the BPH phytotherapeutics and related sterol containing products. The HPLC method to determine β-sitosterol in serum, was evaluated during a pilot study of a clinical trial, to test the bio-equivalence of different phytosterol containing products. The method was found not sensitive enough to determine β-sitosterol in serum, notwithstanding improvements made, Le. changing the extraction ratio; experimenting with higher dosages, and different products. As result, the proposed clinical trial could not be performed, in the future, serum could rather be analysed by gas chromatographic methods. TLC and HPLC analyses of medicinal African potato tea, indicated that it contained hypoxoside, but not β-sitosterol or β-sitosterolin. β-Sitosterol (accepted to be the active of H. hemerocallidea) might not be the main active in African potato tea. Hypoxoside and a compound (red spot compound), noticed on TLC plates of acetone extracts of Prunus africana, Serenoa repens, Moducare®, Harzol®, Immunochoice® and Nutricare®, were extracted with water. This general presence of the red spot compound could point to a possible important function. Preparative TLC was unsuccessful to isolate the red spot compound, but column chromagraphy was successfully applied. From the proton and carbon NMR spectra, it was concluded, that the compound was definitely not a steroid and could either be a coumarin or an isoflavanoid, with a sugar unit (possibly a rhamose) attached to it. Further analyses to elucidate the structure failed due to decomposition of the compound. Further work on structure elucidation is required and possible therapeutic activity should also be investigated. The sterols and sterolins in H. hemerocallidea and related herbal medicine can be qualitatively and quantitatively analysed with the developed TLC and adapted HPLC methods. This provides natural medicine industry with necessary procedures to ensure proper quality, safety and stability. / Dissertation (MSc (Pharmacology))--University of Pretoria, 2006. / Pharmacology / unrestricted

M.Sc. (Biochemistry) / A rich diversity of medicinal plants is found in Southern Africa and approximately 80% of the population still relies on medicinal plants to fulfil its primary health care needs. Many of these medicinal plants are used to treat ailments such as burns, sores, urinary tract infections, colds, flu, rheumatism, gout, cancer, hypertension, diabetes, human immunodeficiency virus infections and acquired immunodeficiency syndrome. An example of such a plant is Hypoxis hemerocallidea (Fisch & CA Mey), formerly known as Hypoxis rooperi and popularly known as the African potato, from the Hypoxidaceae family. This plant is found across five of the South African provinces and corm extracts are reported to contain bioactive compounds that account for the plant’s medicinal and therapeutic properties. This study was conducted to investigate the anti-oesophageal cancer and antimicrobial potential of H. hemerocallidea. In cancer patients, the currently used cancer treatments such as radiotherapy and chemotherapy are ineffective in decreasing disease progression, prolonging survival, providing cure and are associated with side-effects such as cytotoxicity to normal body cells and tumour non-specificity. Therefore, current cancer research is aiming at searching for novel plant-based anticancer compounds that can be used for the development and manufacturing of cancer treatment drugs that will have less side-effects and less toxicity towards the normal human body cells, and ultimately provide cure for cancer. In addition to cancer, infectious diseases still contribute to most premature deaths worldwide and are now becoming more difficult to treat due to multidrug resistance developed by pathogens against many of the currently used antibiotics. This multidrug resistance of human pathogens to antibiotics has led to a search for new antimicrobial compounds from plants sources, for use in the production of new affordable antibiotic drugs to effectively treat infections without posing any unwanted toxicity and harm towards the human body. An oesophageal SNO cancer cell line was treated with H. hemerocallidea extracts and the effect of the extracts on the cancer cells were investigated with cell viability assays (trypan blue dye exclusion and AlamarBlue® viability assays), light microscopy and flow cytometrical analysis (forward and side scatter analysis). The plant extracts were also tested for antimicrobial activities against various microorganisms - Gram-positive and Gram-negative bacteria, yeast and fungi cultures by means of thin layer chromatographic bioautography (TLC-DB), microdilution assays and the BacTiter-GloTM assay. Antimicrobial compounds were then putatively identified and characterised using gas chromatography-mass spectrometry (GC-MS). No morphological changes were observed in the SNO cells and significant cell death did not occur following treatment with either water or ethanolic H. hemerocallidea extracts from fresh or dried corms or leaves. The ethanolic leaf extracts did not show any significant inhibition against any of the microorganisms tested in contrast to the ethanolic extracts from the corms, which showed microbial growth inhibition against Gram-positive bacteria and fungi and partial inhibition of the Gram-negative bacteria. The bioactive compounds responsible for the antibacterial and antifungal activities were identified as levoglucosan (as the major antimicrobial compound), pyrocatechol and hexahydro-3-(2-methylpropyl)-pyrrolo[1,2-α]pyrazine-1,4-dione. These results show that H. hemerocallidea plant extracts possessed no anticancer effects towards the SNO cell line. In addition, the corm extracts of H. hemerocallidea contain a levoglucosan compound, which may work synergistically with other antimicrobial compounds to exert antimicrobial properties. With more research, the antimicrobial compounds in H. hemerocallidea may hold promise for possible candidates for use in the development of antibiotic or antiseptic products (for example, topical creams and lozenges) to be used in the treatment of skin and soft tissue infections caused by bacterial and fungal infections.