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The regeneration of Hypoxis rooperi S. Moore and production of hypoxoside in vitro.Page, Yvonne Margaret. January 1984 (has links)
Against the background of the increasing pharmaceutical importance of
members of the genus Hypoxis L., methods for propagating these plants
and for producing hypoxoside (the believed active compound found within
Hypoxis species) using in vitro techniques, were investigated. These
investigations were accompanied by anatomical observations. Hypoxis
rooperi S. Moore was selected as experimental material because of its
availability and common usage among researchers studying the genus
Hypoxis.
Two aseptic procedures were developed for propagating H. rooperi. These
being the only procedures as yet to be established and documented,
using a member of the family Hypoxidaceae. The first procedure involved
the induction of callus and adventitious shoots from flower bud explants
of H. rooperi . For this response to be initiated, the buds selected for
culture had to be of a specific morphological and physiological age.
The best medium determined for inducing a callusing and shooting
response from these explants, was a MURASHIGE and SKOOG (1962)
medium supplemented with low levels of I-naphthalene acetic acid and
high levels of 6-benzylaminopurine. The rate of this response was
enhanced by the wounding of flower bud explants (i.e. by the excision
of the perianth segments, stamens and style from the buds). Investigations
indicated that callus and adventitious shoot formation was inhibited by
the acropetal positioning of damaged flower buds on the culture medium.
This inhibition was not manifest when buds were placed basipetally or
horizontally on the culture medium. Flower bud harvest time was not
found to have a marked effect upon the numbers of explants responding
in culture. On average 37,5 per cent of the buds formed callus and
adventitious shoots throughout the flowering season.
The subculturing of callus tissue established from H. rooperi flower buds,
onto a MURASHIGE and SKOOG (1962) medium supplemented with the
same hormone levels as were initially used to induce callus and shoot
formation, resulted in the production of multiple adventitious shoots.
Serial subculturing of this tissue indicated that the shoot producing
capacity of the callus, was maintained for at least a year. Shoots produced via this method, when inoculated onto a hormone-free culture
medium, formed roots. Seventy-five per cent of the plantlets regeneratro
in vitro were successfully "hardened-off". Theoretically it was calculated
that using the micropropagation procedure developed, almost 81000 H. rooperi
plantlets could be established from 100 flower bud explants, within a
year.
The second aseptic procedure developed, involved the culturing of explants
excised from the primary thickening meristem region of H. rooperi corms.
The best medium determined for inducing the formation of adventitious
shoots from these explants, was a MURASHIGE and SKOOG ( 1962)
nutrient solution supplemented with: equivalent low concentrations of
I-naphthalene acetic acid and 6-benzylaminopurine; 30 rather than 20
or 40 gl¯¹ sucrose; and 1,0 gl¯¹ casein hydrolysate. Random as opposed
to a basal or side positioning of corm explants upon the culture medium,
resulted in higher numbers of adventitious shoots being produced. The
location of explant excision from within the donor plant was also found
to influence shoot productivity. No significant difference was detected
in the total number of shoots produced from corm explants harvested
at various times of the year.
The rooting of shoots differentiated from corm explants posed few
problems, as most shooted explants eventually formed roots without
being subcultured. Those which did not form roots could be induced to
do so, by the inoculation of the shooted explants onto a culture medium
either devoid of hormones or containing low I-naphthalene acetic acid
levels. Following a rather simple procedure developed, ninety per cent
of the plantlets were "hardened-off". From 100 corm explants it was
therefore possible to regenerate 104 to 112 plantlets within a 3 to 4,5
month period.
Prior to the assessment of the usefulness of in vitro cultures for producing
hypoxoside, qualitative and quantitative techniques for detecting hypoxoside,
were developed. Using these techniques it was established that only the
root-like types of cultured tissue, contained hypoxoside. The levels of
hypoxoside detected within these tissues were much lower than those
found within mature in vivo grown plants. Using the cultured tissue containing the highest levels of hypoxoside, it was shown that the subculturing
of this tissue resulted in a decrease in hypoxoside content. This
effect could be overcome by lowering the levels of nitrogen in the medium
or by culturing the tissue in the dark. These results showed that the
cultured tissue was able to synthesize hypoxoside. To what extent this
synthetic rate can be increased remains very much an academic problem
and one which deserves more attention. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1984.
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An assessment of Hypoxis hemerocallidea extracts, and actives as natural antibiotic, and immune modulation phytotherapies.Muwanga, Catherine January 2006 (has links)
<p>In South Africa, the crude aqueous extract from Hypoxis hemerocallidea is used by AIDS patients to treat opportunistic infections, such as tuberculosis. The rapid emergence of multidrug-resistant tuberculosis, and extreme drug resistant tuberculosis, in recent years, is a major threat to human health. The treatment of TB, nosocomial bacterial infections, and fungal infections is now a clinical challenge, especially in the immuno-compromised individual. There is a dire need for novel antibiotic alternatives with phytotherapies and plant-derived compounds as potentially promising alternatives. The main objective of this study was to investigate the antimycobacterial activity of Hypoxis hemerocallidea, a South African medicinal plant, using Mycobacterium smegmatis.</p>
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Assessment of hypoxoside and its derivatives as anti-cancer drugs.Xulu, Bongiwe Ziphelele. January 2013 (has links)
Extracts of the African potato have long been believed to have anti-cancer properties. The
aim of the current research was to isolate hypoxoside (HYP) from Hypoxis hemerocallidea
(African potato) and synthesize the dimethyl (DMH) and decaacetyl (DAH) derivatives and
to test their selective cytotoxicity on a model consisting of a normal (MCF10A) and
premalignant, invasive breast epithelial cells (MCF10A-NeoT).
Hypoxoside was extracted from the H. hemerocallidea corms using ethanol, purified using a
C-18 reverse phase column and the compound examined by nuclear magnetic resonance
(NMR) spectroscopy and high-resolution mass spectrometry and found to be of high purity.
This was also the case for the synthesized compounds. To assess possible selective effects
(cytotoxicity) of derivatized and underivatized hypoxoside, effects on the metabolism of
premalignant and normal cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Effects on cell
number (total counts) and cell death [trypan blue and propidium iodide (PI) staining for dead
cells versus a lack of staining for live cells] were, thereafter, assessed. Imaging of live
adherent cells was also carried out using acridine orange (AO) and PI for live and dead cells
(respectively). Propidium iodide staining of detached cells was carried out for flow
cytometric determination of cell death (PI indicating early apoptotic or late apoptotic/necrotic
cells).
After treatment of normal (MCF10A) breast epithelial cells and premalignant cHa-rastransfected
(MCF10A-NeoT) derivative breast epithelial cells with HYP, DMH and the DAH
derivative, the MTS assay and the Duncan‟s multiple range, analysis of variance (ANOVA)
post hoc analysis of the MTS results revealed that only the 150 and 300 µM DAH derivative
had a statistically significant effect on the metabolic activity of the abnormal cell line relative
to the dimethyl sulfoxide (DMSO) and revealed no significant effect on the normal MCF-
10A cell line after treatment with any of the test compounds. Supravital PI staining of
adherent cells seemed to indicate a far higher rate of induction of cell death in abnormal cells
than evident in the MTS assay and the PI-based flow cytometry or the trypan blue exclusion
assays and need re-investigating, though result trends were similar.
Total cell counts, show that HYP and its derivatives appear to increase both cancer and
normal cell proliferation significantly, except in the case of DAH at 150 and 300 μM in the
MCF10A-NeoT, without affecting the MCF-10A cell line. The trypan blue method for
detection of cell death, together with total cell counts, the Duncan‟s analysis of MTS results
and a 24 hour exposure to test compounds, seems to constitute an optimal system for drug
screening and indicates the statistically significant selective toxicity of the DAH compound at
150 and 300 μM in the MCF10A-NeoT, suggesting that the DAH derivative at 150 and 300
µM would have significant, selective therapeutic potential on Ras-related malignancies. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.
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An assessment of Hypoxis hemerocallidea extracts, and actives as natural antibiotic, and immune modulation phytotherapies.Muwanga, Catherine January 2006 (has links)
<p>In South Africa, the crude aqueous extract from Hypoxis hemerocallidea is used by AIDS patients to treat opportunistic infections, such as tuberculosis. The rapid emergence of multidrug-resistant tuberculosis, and extreme drug resistant tuberculosis, in recent years, is a major threat to human health. The treatment of TB, nosocomial bacterial infections, and fungal infections is now a clinical challenge, especially in the immuno-compromised individual. There is a dire need for novel antibiotic alternatives with phytotherapies and plant-derived compounds as potentially promising alternatives. The main objective of this study was to investigate the antimycobacterial activity of Hypoxis hemerocallidea, a South African medicinal plant, using Mycobacterium smegmatis.</p>
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An assessment of Hypoxis hemerocallidea extracts, and actives as natural antibiotic, and immune modulation phytotherapiesMuwanga, Catherine January 2006 (has links)
Magister Scientiae - MSc / In South Africa, the crude aqueous extract from Hypoxis hemerocallidea is used by AIDS patients to treat opportunistic infections, such as tuberculosis. The rapid emergence of multidrug-resistant tuberculosis, and extreme drug resistant tuberculosis, in recent years, is a major threat to human health. The treatment of TB, nosocomial bacterial infections, and fungal infections is now a clinical challenge, especially in the immuno-compromised individual. There is a dire need for novel antibiotic alternatives with phytotherapies and plant-derived compounds as potentially promising alternatives. The main objective of this study was to investigate the antimycobacterial activity of Hypoxis hemerocallidea, a South African medicinal plant, using Mycobacterium smegmatis. / South Africa
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African traditional medicines-antiretroviral drug interactions : the effect of African potato (Hypoxis hemerocallidea) on the pharmacokinetics of efavirenz in humans /Mogatle, Seloi. January 2008 (has links)
Thesis (M.Sc. (Pharmacy)) - Rhodes University, 2009.
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African traditional medicines-antiretroviral drug interactions: the effect of African potato (Hypoxis hemerocallidea) on the pharmacokinetics of efavirenz in humansMogatle, Seloi January 2009 (has links)
African Potato (Hypoxis hemerocallidea), (AP) is an African traditional medicine (TM) that is commonly used for various nutritional/medicinal purposes and also by people infected with the human immuno deficiency virus HIV and AIDS patients as an immune booster. The use of AP has also been recommended by the former Minister of Health of South Africa for use by HIV positive people. The main phytochemical component of AP is a norlignan glucoside, hypoxoside, and other relatively minor components have also been reported. A recent in vitro study reported the effects of AP extracts, hypoxoside and rooperol (the metabolite of hypoxoside) on human metabolic enzymes such as the cytochrome P450 (CYP450) group of enzymes and also on the transporter protein, p-glycoprotein (P-gp). This research focussed on investigating the clinical significance of those in vitro effects on the pharmacokinetics of efavirenz (EFV) in humans. EFV was chosen as the substrate drug because it is in first-line regimen of treatment of HIV/AIDS in South Africa, and also has been reported to be a substrate for the specific CYP isozymes, 3A4 and 2B6, in common with APs metabolic involvement with 3A4. A high performance liquid chromatography method with ultra-violet detection (HPLC-UV) for the quantitative determination of EFV in plasma was developed and successfully validated according to international standards with good reproducibility, accuracy, recovery, linear response and requisite sensitivity. The preparation of the plasma samples for analysis was effected by using a simple and rapid precipitation method, and the mobile phase consisted of readily available solvents. EFV in plasma samples was found to be stable under the relevant storage conditions studied. The oral dose of AP, administered as a freshly prepared traditional decoction, was standardised based on the hypoxoside content, and the quality of all the AP decoctions was analysed immediately prior to administration, using a validated HPLC-UV method. A single dose, two-phase sequential study was conducted over a period of 31 days in 10 healthy volunteers. The clinical study was approved by the Rhodes University Ethical Standards Committee, and all the participants agreed to the conditions of the study by giving their informed consent. On day 1 of the study, human subjects were administered a 600 mg EFV tablet and blood samples were collected before dosing and at various intervals over a period of 48 hr post dosing. From day 16, a traditionally prepared AP decoction was administered daily at a standardized dose of 15 mg/kg/day per subject until day 30. On day 29, volunteers were administered a single 600 mg dose of EFV as was done on day 1. Plasma samples were harvested immediately after blood sample collection and frozen at -80 ºC until assayed. Geometric mean ratios of relevant pharmacokinetic parameters, Cmax (maximum plasma concentration achieved following dosing) and AUC0-48 (area under the curve of a plot of drug plasma concentrations versus time representing the extent of absorption) of EFV before and after co-administration of 14 successive daily doses of AP were compared and evaluated to determine whether an interaction had occurred. All subjects completed the study and the geometric mean ratios of Cmax and AUC0-48 were 97.30 and 102.82 with corresponding 90% confidence intervals (CIs) of 78.81-120.14% and 89.04-118.80%, respectively. Whereas the acceptance criteria for the ratios of the AUCs fell within the preset 90% CIs indicating no interaction, the Cmax ratios fell outside the limits. Although the protocol was developed in accordance with the United States of America Food & Drug Administration’s Guidance for Drug Interactions, a priori stating that both criteria need to fall within the acceptance limits to indicate no interaction, an argument is presented to waive the Cmax requirement for the declaration of an interaction. As a result, the pharmacokinetic data generated during this study indicated that the effect of AP on the pharmacokinetics of EFV is not clinically significant. Hence, co-administration of AP is unlikely to affect the clinical use of EFV. In summary the objectives of this project were: 1. To develop and validate a suitable HPLC-UV method for the quantitative determination of EFV in plasma. 2. To perform a mini-validation of the determination of hypoxoside for use as a marker in the quality control and standardisation of AP decoctions. 3. To conduct a clinical interaction study in order to determine whether AP affects the pharmacokinetics of EFV following concurrent administration. 4. To apply the validated HPLC-UV method to determine plasma concentrations of EFV in plasma of human subjects. 5. To use appropriate statistical methods and treatments such as a non-compartmental pharmacokinetic analysis to determine the occurrence of an interaction.
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