Browsing by Author "Stafford, Gary Ivan."

Now showing 1 - 2 of 2

Southern African plants used to treat central nervous system related disorders.
(2009) Stafford, Gary Ivan.; Jäger, Anna Katharina.; Van Staden, Johannes.
The majority of the population in South Africa use traditional health care to treat various mental conditions. This thesis has two main objectives; to bring together a comprehensive and detailed record of psychotropic plants used in southern Africa by indigenous peoples for medicinal or cultural purposes. Secondly, this research attempts to investigate the validity and rationale of the use of these plants by screening them in various biological assays for psychotropic activity. Plants were selected, based on their traditional use and availability, and were screened in four assays, which detect biological activity of a useful nature. A number of in vitro enzymatic and neuronal signal transduction assays were employed in this thesis, the inhibition of the serotonin reuptake transporter protein (SERT); inhibition of catabolic enzymes (e.g. acetylcholinesterase, monoamine oxidase); GABAA- benzodiazepine receptor binding. The influence of legislation, past and present, on the state of traditional medicine is highlighted. Aspects of the philosophies and practises of the various practitioners of South African traditional medicine will be discussed. An annotated list compiled from available ethnobotanical literature of plants traditionally used for central nervous system-related purposes is provided. It contains more than 330 species, from 94 families, which are currently used or have been used for cultural, medicinal and recreational purposes related to the central nervous system (CNS). Where available, information pertaining to plant part used, preparation method, dosage, route of administration, known and potentially active constituents are included. Seventy five extracts from 34 indigenous plant species used in South African traditional medicine or taxonomically related to these were investigated for their affinity to the serotonin reuptake transport protein, making use of an in vitro [3H]-citalopram serotonin reuptake transport protein binding assay. Aqueous and 70% ethanolic extracts of various plant parts were screened and 45 extracts derived from 15 plant species showed affinity. The affinity of 12 extracts from four plants was characterized as high (more than 50% inhibition at 5, 1, and 0.5 mg/ml). Plant species with high affinity to the serotonin reuptake transport protein included Agapanthus campanulatus, Boophone disticha, Datura ferox and Xysmalobium undulatum. Agapanthus campanulatus yielded high activity in aqueous extracts from leaves and flowers. B. disticha showed high activity both in aqueous and ethanolic extracts of leaves and bulbs. D. ferox showed high activity in aqueous extracts from the seeds and X. undulatum showed high activity in the ethanolic extract of the whole plant. Two compounds, buphanadrine and buphanamine, were isolated by bioassay-guided fractionation on vacuum-liquid-chromatography (VLC) and preparative thin-layer-chromatography (TLC) from B. disticha. The structures of the compounds were determined by 1H and 13C NMR. Fractions were tested for affinity to the serotonin transporter in a binding assay using [3H]-citalopram as a ligand. The IC50 values of buphanidrine and buphanamine were 274 ìM (Ki = 132 ìM) and 1799 ìM (Ki = 868 ìM), respectively. The two alkaloids were also tested for affinity to the 5HT1A receptor, but only showed slight affinity. Aqueous and ethanol extracts of 43 plants that are traditionally used to treat against epilepsy and convulsions were initially tested in the GABAA-benzodiazepine receptor binding assay, where the binding of 3H-Ro 15-1788 (flumazenil) to the benzodiazepine site is measured. The GABAA-benzodiazepine receptor complex is involved in epilepsy and convulsions. Out of the 118 extracts tested, one aqueous and 18 ethanol extracts showed activity. The most active extracts were the ethanolic leaf extracts of Searsia tridentata, Searsia rehmanniana and Hoslundia opposita and the ethanolic corm extract of Hypoxis colchicifolia, which all showed good dose-dependent activity. A further forty-six ethanol extracts from another 35 species, both indigenous and exotic that are traditionally used predominantly as sedatives or to treat various CNS-related ailments were tested in the GABAA-benzodiazepine receptor-binding assay. Out of the 46 extracts tested, seven showed good activity and 10 showed moderate activity. The most active extracts were the ethanolic leaf extracts of Arctopus echinatus, Artemisa afra, four Helichrysum species and Mentha aquatica which all showed good dose-dependent activity. Two biflavonoids with activity in the 3H-Ro 15-1788 (flumazenil) binding assay were isolated by high pressure liquid chromatography (HPLC) fractionation of the ethanol extract of the leaves from Searsia pyroides. The structures of the two biflavonoids were elucidated by nuclear magnetic resonance spectroscopy (NMR) to be agathisflavone and amentoflavone. Agathisflavone and amentoflavone competitively inhibited the binding of 3H-Ro 15-1788 with a Ki of 28 and 37 nM, respectively. Extracts of Searsia dentata and Searsia pentheri were not as active as the extract from Searsia pyroides; both were found to contain apigenin and agathisflavone. The monomer apigenin, agathisflavone and amentoflavone were fitted into a pharmacophore model for ligands binding to the GABAA receptor benzodiazepine site. This reflected the affinities of the compounds in the [3H]-flumazenil binding assay. Mentha aquatica, a mint that is found in Europe and Africa, is used in Zulu traditional medicine for spiritual purposes. The ethanolic leaf extract showed a strong affinity to the GABA-benzodiazepine receptor. Viridiflorol from the essential oil and (S)-naringenin from an ethanolic extract was isolated by bioassay-guided fractionation using binding to the GABA-benzodiazepine site. Viridiflorol had an IC50 of 0.19 M and (S)-naringenin of 0.0026 M. Twenty plants used in Zulu traditional medicine for several CNS-related ailments were screened for MAO inhibition and specific MAO-B inhibition activity. MAO-B inhibitors are currently employed in the treatment of neurodegenerative related illnesses such as Parkinson's and Alzheimer's diseases. A photometric peroxidase linked assay was used to determine the inhibition of the oxidative deamination of tyramine by MAO isolated from rat liver. Ruta graveolens exhibited the best MAO inhibitory activity (ethyl acetate leaf extract = IC50 5 ± 1 ìg/ml, petroleum ether extract = 3 ± 1 ìg/ml) and specific MAO-B inhibition (ethyl acetate leaf extract = IC50 7 ± 6 ìg/ml petroleum ether extract = 3 ± 1 ìg/ml). Schotia brachypetala, Mentha aquatica and Gasteria croucheri also exhibited good MAO-B inhibition activity. Six extracts of varying polarity of Mentha aquatica were tested in a photometric peroxidase linked MAO bioassay. The 70% ethanol extract had highest inhibitory activity. (S)-Naringenin was isolated from the extract by bioassay guided fractionation on VLC and preparative TLC. The structure of the compound was determined by 1H, 13C and 13C-DEPT NMR and optical rotation. The IC50 values for MAO inhibition by naringenin were 342 ± 33 ìM for the rat liver mitochondrial fraction, 955 ± 129 ìM for MAO-A and 288 ± 18 ìM for MAO-B respectively. South African traditional medicine clearly utilizes many botanical species with CNS-related activity. Only a small number of the more than 330 southern African plant species reported to treat or alter the CNS have been scientifically evaluated. To date very few of the active compounds have been isolated and identified.
Storage of frequently used traditional South African medicinal plants.
(2003) Stafford, Gary Ivan.; Jäger, Anna Katharina.; Van Staden, Johannes.
The post-harvest physiology of nine frequently used indigenous southern African medicinal plants was investigated, in particular the effects of storage time and accelerated ageing on the biological activity and chemical constituents of these plants. Water, ethanol and hexane extracts of fresh plant material as well as material that had been stored in dry form in paper bags at room temperature for 90 days (short-term) were tested. Three bioassays, the COX-1 anti-inflammatory assay, nematode anthelmintic assay and minimum inhibitory concentration anti-bacterial assay, were used to determine biological activity. Thin layer chromatography of all the plant extracts were used to determine changes in chemical composition. The plants tested were Alepidea amatymbica Eckl. & Zeyh., Leonotis leonurus (L.) R. Br., Drimia robusta Bak., Vernonia colorata (Willd.) Drake, Scilla natalensis Planch., Eucomis autumnalis (Mill.) Chitt. subsp. autumnalis, Bowiea volubilis Harv. ex Hook. f., Helichrysum cymosum (L.) D. Don and Siphonochilus aethiopicus (Schweinf.) B. L. Burtt. Only those plants, which are known to exhibit a particular biological activity either traditionally or scientifically, were tested in the relevant bioassays. Of the plant extracts tested for anthelmintic activity only the water extracts showed activity and very little change in activity was observed after storage. Of the plant extracts tested for anti-inflammatory activity the ethanol extracts generally yielded highest activity. S. natalensis and B. volubilis both showed an increase in cyclooxygenase inhibition (anti-inflammatory) activity after storage whereas S. aethiopicus, H. cymosum, D. robusta and V. colorata showed a loss in activity after storage. The anti-inflammatory activity of E. autumnalis did not change. The water extracts of plants tested for antibacterial activity showed no activity, whereas the ethanol extracts generally showed an increase in activity. The TLC fingerprints indicated that there was chemical break-down during storage in certain species. These corresponded to the changes in biological activity. Alepidea amatymbica, Eucomis autumnalis, Helichrysum cymosum, Leonotis leonurus, Siphonochilus aethiopicus and Vernonia colorata were investigated further as to the effect of one year's storage (long-term storage) on their chemical composition and biological activity. Similar trends to that of the 90-day storage were observed. Activity gained in plants that were stored for 90 days was retained after a year of storage. Elevated temperature and humidity (55 C and 100% relative humidity) were used to accelerate the ageing process of Alepidea amatymbica, Leonotis leonurus and Vernonia colorata plant material. Again changes in the chemical composition and biological activity were observed, and the extent of these changes was greater than those in the stored material. The compounds responsible for the cyclooxygenase inhibition in the ethanolic extracts of Alepidea amatymbica leaf material appear to be stable and were not affected by the conditions of the accelerated ageing procedure (55 C and 100% humidity for seven days), but the root material lost activity, as did the leaf material of Leonotis leonurus. The leaf material of Vernonia colorata showed a slight (8%) increase in cyclooxygenase inhibition activity. The response of the plant material to accelerated ageing with respect to antibacterial activity varied with plant species. Alepidea amatymbica root material and Vernonia colorata leaf material appear to be stable whereas the other plant materials lost activity after prolonged (25 days) ageing.