University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

D. Tech. Pharmaceutical Sciences. / Aims of the research project was to develop and optimise rapid quality control methods for H. gordonii raw material and products. The second aim was to determine whether the perceived active component of H. gordonii (P57) is transported across porcine intestinal and buccal mucosa.

Thesis (MTech. degree in Pharmaceutical Sciences)--Tshwane University of Technology, 2009. / Quality control procedures are vital in the pharmaceutical industry to guarantee the authenticity and quality of products. A major challenge in quality assurance of herbal material is the vast variation of active constituents in plants from the same species. As a result of this variation, the selection of only a few compounds as criteria for quality control is inadequate. Pelargonium (P.) sidoides is indigenous to South Africa and highly valued by traditional healers as a remedy to treat coughs, upper respiratory tract irritations and gastrointestinal conditions. An ethanolic extract of P. sidoides is used in the proprietary herbal tincture known as Umckaloabo®. The composition and concentration of polyphenols are parameters which determine the quality of this herbal medicine because it provides several therapeutic benefits in the non-specific medicinal treatment of infectious diseases. Despite the commercial development of P. sidoides very few studies have been conducted to document the full phytochemical range of variation for natural populations and no study has been published on the development of a fast accurate quality control method for the validation of raw material.

The results of the investigation of Perilla frutescens (L.) Britton growth, vegetation rhythmic dependence upon meteorological factors is given here. Experiments were carried out in 2001 – 2005 during vegetation period at Kaunas Botanical Garden of Vytautas Magnus University. Object of investigations - Perilla frutescens (L.) Britton a medicinal, herbal immunomodulator, annual herbaceous deadnettle family (Lamiaceae Lindl.) plant originated from Asia passes all development cycle in middle Lithuanian climatic conditions. P. Frutescens vegetation length on the average is 167 days. Earliest initiation of vegetation and optimal climatic conditions for growth are when HTC=1,60-1,80, that is excess heavy humidity. Optimal climatic conditions for massive flowering and seed ripeness are when HTC=1,20, then massive flowering starts 10 days earlier and Lasts for 25 days. Strong correlation interdependence was established between massive flowering and HTC (R2=0,9406). After the investigation of P.frutescens terraneous parts (high, relative plain area) consistent patterns of vegetation rhythmic, the optimal harvest time, which determines raw material’s quality and quantity, was established, by using mathematical – statistical methods.

This thesis investigates the possibility of using aposematic insects as guides to plants that contain pharmacologically-active compounds. Plants were monitored within national parks in the Republic of Panama over a period of six months and all insects feeding on them were collected and raised in captivity. The insects were then extracted and analyzed to determine how they were treating toxic chemical compounds in their host plant. Two principal plants were investigated with their associated insects: (1) Vismia baccifera and (2) Mikania guaco. One generalist and one specialist Lepidopteran species were found to sequester vismione B from their host plant Vismia baccifera, a cytotoxic compound active against three cancer cell lines. Two specialist Coleopterans were found to sequester the novel compound Guacanone, isolated by the primary author from the vine Mikania guaco and active against Trypanosoma cruzi, the causative agent of Chagas' disease. A generalist Coleopteran was found to not sequester this compound. (Abstract shortened by UMI.)

The aim of this study was to investigate the possible toxicity of the flavonoid-containing plant, Artemisia afra and especially establish the safety of the aqueous extract of this plant after acute and chronic administration to mice and rats respectively.

The Orchidaceae makes up the largest and most diverse family of flowering plants. Orchids are popular, often expensive ornamentals, with a broad range of ethnobotanical applications. There is very limited documented information on South African medicinal orchid species; no formal pharmacopoeia outlining ethnobotanical uses; and ethnobotanical and distribution records are either scarce or inconsistent and plant populations are becoming gradually smaller. There have been significant developments in medicinal orchid research worldwide with medicinal use and corresponding pharmacological and phytochemical properties being extensively investigated. It is evident from the literature that there is no pharmacological research on South African medicinal orchids; hence the need to explore biological activity and chemical composition of South African medicinal orchid species. The ethnobotanical approach used to select the orchid species for pharmacological and phytochemical research elsewhere, yielded valuable biological compounds. Thus, a similar approach was applied to South African medicinal orchids. There are approximately 20 000 species and 796 genera of orchids distributed across the world. In southern Africa, orchids are widely represented with 55 genera and 494 species. Approximately 75% are endemic to this region. As part of the current investigation a review of available ethnobotanical literature on South African medicinal orchids was prepared. The review revealed that an estimated 49 indigenous orchid species from 20 orchid genera are currently being informally traded and used in South African traditional medicine. They are used primarily for medicinal and cultural purposes, especially by the Zulu community in South Africa. Medicinal uses of orchid species include: treatment of inflammatory, intestinal, neurological and reproductive disorders and emetics are used to cause emesis. Non-medicinal uses of orchid species include: love, fertility, protective and lethal charms. Based on their ethnobotanical uses and endemism, South African orchids were considered to be one of the untapped sources of bioactive compounds that needed to be researched. The current investigation addressed the broader aims of medicinal plant research by determining the efficacy, safety and chemical profile of seven indigenous orchid species used in South African traditional medicine and practices. The biological and toxic effects of orchid plant extracts were assessed using established pharmacological bioassays. The phytochemical evaluation of the seven orchid plant extracts provided insight into the classes of chemical compounds present and their possible role in the observed biological activities. The potential of plant extracts from seven orchid species used in South African traditional medicine, as sources of natural bioactive products, are discussed. The current investigation determined the biological activity and chemical profile of seven orchid species commonly traded in KwaZulu-Natal herbal markets: Ansellia africana Lindl., Bulbophyllum scaberulum (Rolfe) Bolus, Cyrtorchis arcuata (Lindl.) Schltr., Eulophia hereroensis Schltr., Eulophia petersii (Rchb.f.) Rchb.f., Polystachya pubescens (Lindl.) Rchb.f. and Tridactyle tridentata (Harv.) Schltr. Well established in vitro micro-dilution bioassays were used to determine the antibacterial, antifungal, anthelmintic activities of crude orchid extracts. A minimum inhibitory and/or lethal effect of organic and aqueous crude orchid extracts was observed against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Candida albicans and Caenorhabditis elegans. Tridactyle tridentata aqueous root extract produced the most effective antibacterial activity against S. aureus (0.049 mg/ml). All T. tridentata organic root extracts produced significant inhibitory activities against B. subtilis and S. aureus. Eulophia petersii DCM pseudobulb extracts significantly inhibited all bacterial strains tested (0.39 mg/ml against S. aureus and 0.78 mg/ml against B. subtilis, E. coli, and K. pneumoniae). Eulophia hereroensis 80% EtOH root extract was the only other extract to exhibit significant inhibitory effects against K. pneumoniae (0.65 mg/ml). After 48 h C. albicans was most susceptible to P. pubescens aqueous pseudobulb extract (0.0816 mg/ml). Eulophia petersii DCM pseudobulb extract however, exhibited significant activity against C. albicans (0.65 mg/ml) over 72 h. Cyrtorchis arcuata leaf and root extracts were the most effective anthelmintic extracts with MLCs of 0.041 mg/ml for 80% EtOH leaf and root extracts; 0.049 mg/ml for aqueous leaf extracts and 0.78 mg/ml for aqueous and DCM root extracts. Caenorhabditis elegans was most susceptible to all A. africana and T. tridentata organic root extracts. A similar significant effect was observed for all E. petersii organic pseudobulb extracts, DCM extracts and organic root extracts of B. scaberulum. Only the DCM tuber and root extracts of E. hereroensis exhibited lethal effects on C. elegans. All of the P. pubescens extracts showed poor anthelmintic activity. Similarly, in vitro enzyme based cyclooxygenase (COX) 1 and 2 and acetylcholinesterase (AChE) inhibitory bioassays, revealed significant inhibition of COX-1, COX-2 and AChE enzymes by crude organic and certain aqueous orchid extracts. Out of a total of 53 evaluated extracts, 21 and 13 extracts exhibited significant anti-inflammatory activity in the COX-1 and COX-2 assays respectively. The DCM tuber extract of E. hereroensis was the only extract to significantly inhibit both COX enzymes, 100.02±0.11% and 87.97±8.38% respectively. All B. scaberulum root extracts (DCM, EtOH and water) exhibited COX-2 selective inhibitory activity (100.06±0.01, 93.31±2.33 and 58.09±3.25%). Overall, the DCM root extract of A. africana was found to be the most potent extract (EC50 0.25±0.10 mg/ml). The 80% EtOH root extract of B. scaberulum was the most potent in the COX-2 assay (EC50 0.44±0.32 mg/ml). Generally the root extracts exhibited greater AChE inhibitory activity; where the most active extract was B. scaberulum DCM root extract (EC50 0.02±0.00 mg/ml). All aqueous extracts, except that of A. africana roots and B. scaberulum pseudobulbs, showed poor or no COX-1 and COX-2 inhibition. The antioxidant capacity of crude orchid extracts was determined using: hydrogen atom transfer (HAT) (β-carotene/linoleic acid assay) and single electron transfer (SET) (2,2‟-diphenylpicrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing antioxidant power (FRAP) assay) reaction-based assays. Potent antioxidant effects were observed for certain crude methanolic orchid extracts. Generally, there was a dose-dependent change in radical scavenging activities of crude extracts from which EC50 values were determined. The root extracts of all species, except that of E. petersii, had consistently more effective radical scavenging activity than that of other plant parts within each species. The pseudobulb extract of E. petersii, was the most potent extract (EC50 1.32±0.86 mg/ml). In the β-carotene-linoleic acid assay, based on the oxidation rate ratio (ORR), the leaf extract of T. tridentata and the root extracts of C. arcuata and E. hereroensis exhibited the best antioxidant effects (0.02, 0.023 and -0.15 respectively). Similarly, the average antioxidant activity (%ANT) of these samples was greater than that of BHT (95.88±6.90%) and all other samples. Bulbophyllum scaberulum leaf, pseudobulb and root extracts, E. petersii pseudobulb extract and T. tridentata root extract also exhibited a greater capacity to prevent β-carotene oxidation when compared to BHT. All crude orchid extracts tested demonstrated a general dose-dependent response in the ferric reducing power assay. The reducing power of ascorbic acid (0.08 mM) and BHT (0.05 mM), as measured as absorbance, was 1.12±0.12 and 0.73±0.08 respectively. At 6.25 mg/ml, A. africana root and E. petersii pseudobulb extracts were the most effective in reducing power activity. The short-term bacterial reverse mutation Ames Salmonella/microsome mutagenicity (ASMM) assay, which makes use of mutant histidine-dependent Salmonella typhimurium strains, was used to determine the mutagenicity and toxicity of crude orchid extracts. In the presence of a mutagen S. typhimurium TA98 strain detects frameshift events while the TA100 and TA102 strains detect base-pair substitutions. In the absence of metabolic activation, mutagenic extracts were observed against the TA98 strain only. All A. africana DCM leaf and stem extracts tested, the DCM root extract (0.5, 0.05 mg/ml) and EtOH leaf, stem and root extracts at 5 mg/ml exhibited mutagenic effects. The EtOH root extracts (5, 0.5 mg/ml) of B. scaberulum exhibited mutagenic indices (MI) comparable to that of 4NQO (17.00 and 13.00, respectively). Eulophia petersii PE pseudobulb extract demonstrated mutagenic potential at 5 mg/ml. The ethanolic root extracts of T. tridentata showed mutagenic effects at 5 and 0.5 mg/ml. The mutagenicity index (MI) with metabolic activation (S9) was determined using only the TA98 strain; where no mutagenic effects were observed. In the phytochemical evaluation of crude methanol orchid extracts, the Folin-Ciocalteu assay for total phenolics, butanol-HCl assay for condensed tannins, rhodanine assay for gallotannins and vanillin assay for flavonoids revealed a quantitative chemical profile of the tested samples. The correlation between observed biological effects and chemical compounds present was found to be generally significant. The significant antimicrobial, anthelmintic, anti-inflammatory and antioxidant activity of E. petersii pseudobulb extracts and E. hereroensis tuber and root extracts may be attributed to their high total phenolic content. Alternatively, the significant levels of gallotannin content in E. hereroensis may have contributed to the bioactivity. The flavonoid content of B. scaberulum and T. tridentata may explain the potent activity observed in the anti-inflammatory, antioxidant and acetylcholinesterase inhibitory assays; while the flavonoid content C. arcuata may have contributed to the potent anthelmintic and antioxidant activities. The significantly higher levels of gallotannin content may explain the significant anti-inflammatory and anthelmintic activity of A. africana. A number of biologically active compounds have been isolated from certain Orchidaceae species around the world on the basis of their traditional medicinal uses. The traditional uses of these orchid species were scientifically validated. No pharmacological research has been previously conducted on South African medicinal orchids; therefore the current investigation has produced novel findings on the efficacy and safety of these orchid species and promotes the continued research of medicinal orchids in South Africa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Ingestion of extracts prepared from various medicinal plants to induce or augment labour is common amongst Black South African women during the late stages of pregnancy. This applies particularly to the rural areas where modern health care facilities are often lacking. Many of these plants have not been investigated scientifically and one needs to substantiate claims of quality, safety and efficacy. Furthermore, it is believed that the consumption of these plant extracts can result in foetal meconium staining at delivery. An investigation into the uterotonic properties of three plants viz. Ekebergia capensis Sparrm. Clivia miniata (Lindl.) Regel. and Grewia occidentalis L. were carried out using guinea pig uterine smooth muscle in vitro. Supercritical fluid extraction was performed with water modified supercritical carbon dioxide to extract the uterotonic components. An attempt was also made to couple supercritical fluid extraction directly on-line to the bioassay so that on line screening of crude plant extracts could be performed within short periods of time. The effects of supercritical CO2 decompression on temperature and pH of the muscle bathing solution were considered since these factors affect muscle contractility. The direct effects of excess CO2 on intracellular mechanisms were eliminated by constructing a CO2 reduction interface together with passage of carbogen which aided in the rapid displacement of excess CO2, As samples of these extracts were found to induce muscle contraction, supercritical fluid fractionation (SFF) was performed by sequentially increasing the fluid density. Extracted fractions were obtained by sequentially increasing the pressure at constant temperature and modifier concentration in an attempt to identify the active fractions. Extractions were performed at 200 atm, 300 atm and 400 atm respectively. Subsequent testing of these fractions enabled the detection of active and inactive fractions as well as a fraction that had a spasmolytic effect on uterine muscle. The 400 atm extracts of E. capensis and C. miniata displayed maximum activity while only the 300 atm extract of G. occidentalis induced uterine muscle contraction. Subsequent analysis of the sequentially extracted fractions, by high performance liquid chromatography and micellar electrokinetic capillary chromatography revealed that certain compounds present in the fractions that stimulated muscle contraction, were sensitive to the extraction pressure hence making it possible to determine the compounds that were likely to be active. Column chromatography followed by various spectroscopic techniques were performed in an attempt to isolate and elucidate the structures of the compounds that were present in the plant extracts. The extract of Ekebergia capensis yielded five known compounds (B-sitosterol, oleanonic acid, 3-epioleanolic acid, 2,3,22,23-tetrahydroxy-2,6,1 0, 15,19 ,23-hexamethyl-6, 10, 14, 18- tetracosatetrene and 7-hydroxy-6-methoxy coumarin. The extract of Clivia miniata yieded linoleic acid and 5-hydroxymethyl-2-furancarboxaldehyde while the extract of Grewia occidentalis yielded 3-(4-hydroxy-3-methoxyphenyl)-2-propenal, a novel compound 2,2' ,6,6'-tetramethoxy-4'-al-4-(w-oxo-E-propenyl)-biphenyl and oleanonic acid. The pure compounds were further evaluated pharmacologically to identify the active components and assess the physiological mode of action by the use of various receptor blockers. Oleanonic acid, 3-epioleanolic acid, linoleic acid and 5- hydroxymethyl-2-furancarboxaldehyde and 3-(4-hydroxy-3-methoxyphenyl)-2-propenal were found to induce an agonistic muscle response. All these compounds were observed to mediate their effects through the cholinergic receptors. The results obtained in this study supports the claim of these plants possessing uterotonic properties. / Thesis (Ph.D.)-University of Natal, Durban, 1997.

Tulbaghia (Alliaceae) is a genus of plants with medicinal, ornamental and nutritive value. Different plant parts such as roots, bulbs, leaves and flowers are used in the treatment of a variety of conditions. The bulbs of Tulbaghia violacea are used as a remedy for pulmonary tuberculosis as well as an anthelmintic. Due to the extensive harvesting of plants in this genus, the genus is susceptible to overexploitation and may eventually become threatened with extinction. It was therefore the aim of this study to systematically examine the micropropagation of Tulbaghia ludwigiana and Tulbaghia violacea, as well as to evaluate the antimicrobial and phytochemical properties of micropropagated plants. Seeds of T. ludwigiana and T. violacea were successfully decontaminated using 70% ethanol, 1% Benlate and 3.5% NaOCl. Temperature played a significant role in the germination of both species while light did not play a significant role in this process. Light did not play a significant role in the stomatal density of T. violacea seedlings. Hypocotyls were the regenerative part of the seedlings in both species. A low number of shoots was yielded by the combination of various concentrations of NAA and mTR in the growth medium in both species. There were more isoprenoid cytokinins than there were aromatic cytokinins in each of the seedling sections of T. violacea that were analysed, with tZ being the predominant isoprenoid cytokinin, while BA was the predominant aromatic cytokinin. Shoots of both species were successfully rooted in a medium with IBA, while some shoots had simultaneously rooted during shoot multiplication. Potting soil and vermiculite were used in the acclimatization of both species, where T. violacea plantlets acclimatized successfully. This was not the case for T. ludwigiana. Micropropagated T. violacea plantlets contained higher concentrations of phytochemical compounds and displayed better antibacterial activity than outdoor-grown plants. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.