Many therapies prescribed by traditional healers in Southern Africa include plant combinations to treat infectious diseases. Artemisia afra is one of the most commonly used traditional medicines in African traditional medicine and most often given in combination with other plants. This plant‟s popularity coupled with its wide range of uses in combination serves as the rationale for the bases of this study. In this study, combinations of A. afra (essential oils and plant extracts), which are commonly used for the treatment of respiratory diseases were studied from an antimicrobial perspective in order to determine if a scientific basis exists for their combined use. The plants used often in double or triple combination with A. afra in the treatment of respiratory tract infections are Lippia javanica, Osmitopsis asteriscoides, Agathosma betulina, Eucalyptus globulus, Allium sativum, Leonotis randii, Tetradenia riparia and Zanthoxylum capense. Essential oils from plant samples were analysed using gas chromatography coupled to mass spectroscopy (GC-MS). Compounds found in highest concentrations were camphor (41.0%) in A. afra, linalool (70.7%) in L. javanica, 1,8-cineole (59.0%) in O. asteriscoides, isomenthone (31.4%) in A. betulina, 1,8-cineole (63.0%) in E. globulus and β-caryophyllene (32.4%) in T. riparia. Dichloromethane: methanol extracts and aqueous extracts were prepared for each plant using the dried ground plant material collected. The antimicrobial activities of each sample as well as each combination (including essential oils) were tested using the minimum inhibitory concentrations (MIC) assay against a panel of respiratory tract organisms. The highest sensitivities observed for the essential oils were that of E. globulus against Cryptococcus neoformans with a MIC value of 0.6 mg/ml. The dichloromethane: methanol extracts showed the most activity with E. globulus against Moraxella catarrhalis (MIC value of 0.01 mg/ml). The aqueous extracts showed the best activity with Z. capense against Streptococus agalactiae with a MIC value of 0.4 mg/ml. The 1:1 fractional inhibitory concentration (ΣFIC) values of the combinations of A. afra with L. javanica, A. afra with O. asteriscoides, A. afra with A. betulina, A. afra with E. globulus and A. afra with Z. capense were calculated from the MIC data. Synergy, additivity, indifference and antagonistic interactions within the combinations were then interpreted. The most significant interactions of the double combinations with synergistic ΣFIC values of 0.2 were the combination of the dichloromethane: methanol extracts of A. afra with O. asteriscoides against Streptococcus pyogenes and the combination of the aqueous extracts of A. afra with E. globulus against Streptococcus pneumoniae. Significant antagonism was noted with the combination of the dichloromethane: methanol extracts of A. afra with E. globulus against Enterococcus faecalis. The ΣFIC results of the combinations of A. afra with L. javanica, O. asteriscoides, A. betulina, E. globulus or with Z. capense were used to calculate ratios and plotted on to an isobologram. The isobolograms were interpreted with regard to any synergy, antagonism, or additive interactions present in the combination. Isobolograms revealed the most significant activity with the combination of the aqueous extracts of A. afra with E. globulus against C. neoformans with all the ratios tested being synergistic. The most prominent antagonism (five ratios) noted was in the combination (dichloromethane: methanol extracts) of A. afra with E. globulus against M. catarrhalis. The triple combinations analyzed for their antimicrobial activity were the combinations of A. afra with O. asteriscoides and E. globulus, A. afra with L. randii and E. globulus, A. afra with A. sativum and Z. capense and the combination of A. afra with T. riparia and salt. The most significant synergistic activity was noted for the combination of the essential oils A. afra with T. riparia and salt against Mycobacterium smegmatis with a ΣFIC value of 0.2. The combination of A. afra with O. asteriscoides and E. globulus of (dichloromethane: methanol extracts) displayed the most antagonistic activity against M. catarrhalis. When analysing the combinations that include A. afra, it was noted that adjuncts are an important combination ingredient in the traditional method of preparation. These were also tested for their activity. The combinations that include adjuncts i.e. honey, salt, vinegar, brandy and milk showed mainly indifferent interactions. This indifference noted supports the use of these adjuncts by traditional healers as it serves to verify that these adjuncts are at least not hindering the activity of the plant itself, which is a positive direction for future investigations. Traditional medicine, with regard to A. afra, as prescribed by traditional healers, has commonly employed the use of combinations of more than one plant to treat respiratory conditions. When the antimicrobial activities in combination were examined from a scientific viewpoint, there is evidence of some bases for their traditional use. The results obtained from the testing of the essential oils validate its traditional use as an inhalant. The dichloromethane: methanol extracts showed results varying from synergy to antagonism while the aqueous extracts showed good antimicrobial activity. It is recommended that future studies should be conducted into these interactions to determine the benefits of these combinations for possible use in the commercial and primary health-care sectors.

Aim of the study: In traditional healing, the burning of selected indigenous medicinal plants and the inhalation of the liberated smoke are widely accepted and a practiced route of administration. This study elucidated the rationale behind this commonly practiced treatment by examining the antimicrobial activity for five indigenous South African medicinal plants commonly administered through inhalation (Artemisia afra, Heteropyxis natalensis, Myrothamnus flabellifolius, Pellaea calomelanos and Tarchonanthus camphoratus). Material and Methods: An apparatus was designed to simulate the burning process that occurs in a traditional setting and the smoke fraction was captured for analysis and bioassay. Methanol and acetone extracts as well as the essential oil (for the aromatic species) were prepared and assayed in parallel with the smoke fraction. Results: Antimicrobial data revealed that in most cases, the ‘smoke-extract’ obtained after burning had lower minimum inhibitory concentration (MIC) values than the corresponding solvent extracts and essential oils. The combustion, acetone and methanol extracts produced different chromatographic profiles as demonstrated for Pellaea calomelanos where several compounds noted in the smoke fraction were not present in the other extracts. Conclusion: These results suggest that the combustion process produces an ‘extract’ with superior antimicrobial activity and provides in vitro evidence for inhalation of medicinal smoke as an efficient mode of administration in traditional healing.

The phytochemical investigation of an ethanol extract of Artemisia afra, led to the isolation of six known compounds, Acacetin (1) 12α,4α-dihydroxybishopsolicepolide (2), Scopoletin (3) α-amyrin (4), Phytol (5) and a pentacyclic tri-terpenoid Betulinic acid (6). The isolated compounds were evaluated for their anti-microbial activity against Gram positive (Actinomyces naeslundii, Actinomyces israelii and Streptococcus mutans), Gram negative bacteria (Privotella intermedia, Porphyromonus gingivalis and Aggregatibacter actinomycetemcomitans previously known as Actinobacillus actinomycetemcomitans) and Candida albicans. The crude extract of A. afra inhibited the growth of all tested microbial species at concentration range of 1.6 mg/ml to 25.0 mg/ml. The compounds 1-6 also showed activity range at 1.0 mg/ml to 0.25 mg/ml. Three best compounds which showed good activity were selected for further studies. Cytotoxicity of the extract and compounds was determined using the XTT (Sodium 3’-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate) cell proliferation kit. The antioxidant activity of the extract and compounds was done using the DPPH scavenging method. The extract showed good antioxidant activity with an IC50 value of 22.2 μg/ml. Scopoletin had a strong transformation of the DPPH radical into its reduced form, with an IC50 value of 1.24 μg/ml which was significant to that of vitamin C (1.22 μg/ml). Acacetin and Betulinic acid exhibited a decreased scavenging activity with the IC50 of 2.39 and 2.42 _g/ml, respectively. The extract and compounds showed moderate toxicity on McCoy fibroblast cell line and the extract influenced the release of cytokine against Hep2 cells. Scopoletin was relatively non-toxic with an IC50 value of 132.5 μg/ml. Acacetin and betulinic acid also showed a smooth trend of non-toxic effects at lower concentrations and toxic at higher concentrations with IC50 values of 35.44 and 30.96 μg/ml. The obtained results in this confirmed the use of A. afra in the treatment of microbial infections. / Dissertation (MSc)--University of Pretoria, 2012. / Plant Science / unrestricted

Magister Pharmaceuticae - MPharm / Artemisia afra (A. afra) is traditionally used for a variety of ailments and contain flavonoids e.g. luteolin which may contribute to some of its activity. It is generally administered as a tea or decoction, and such liquid dosage forms present challenges as far as long term storage and stability are concerned, as well as sub-optimal oral bioavailability of actives they contain. Freeze dried aqueous extracts (FDAE) can alleviate such problems but may be hygroscopic and unstable. The use of modified forms of FDAE can counter the problem of hygroscopicity (e.g. use of alginate) and alleviate the issue of sub-optimal bioavailability of plant actives (e.g. polymethylmethacrylate). The objectives of this study, were to: (1) prepare the freeze dried aqueous extract (FDAE) and modified forms, which include alginate-extract beads (alginate-FDAE) and polymethylmethacrylate coated alginate matrix beads of herbal extract (PMMA-alginate-FDAE) of the FDAE of A. afra, (2) determine and compare the pharmaceutical characteristics of the above mentioned preparations of A. afra,(3) quantify and compare the total flavonoid and specifically luteolin levels of the different forms of A. afra,(4) evaluate and compare the release characteristics of FDAE of A. afra from the alginate-FDAE and PMMA-alginate-FDAE beads in gastrointestinal fluids and (5) determine the intestinal permeability of luteolin contained in selected modified Artemisia afra extract preparations. It was hypothesized that making the alginate beads and the polymethylmethacrylate coated alginate beads would make the FDAE less hygroscopic with a lower moisture content, that the rate of release of luteolin from A. afra FDAE into gastrointestinal fluids would be faster than from the modified forms, and that the effective gastrointestinal permeability of luteolin in the alginate-FDAE and PMMA-alginate-FDAE beads of A. afra is equal to that in FDAE. To realize these objectives, the FDAE was prepared by freeze drying the aqueous extract of the A. afra dried leaves, alginate-FDAE prepared by dispersing FDAE into 4% sodium alginate solution, then adding the resulting stock solution into a 2% calcium chloride solution and drying resulting beads and PMMA-alginate-FDAE prepared by a modified water-in-oil-in-water emulsion solvent evaporation method using water as an internal aqueous phase. Using pharmacopoeial methods and methods adapted from other workers the organoleptic and pharmaceutical characteristics were determined to compare the pharmaceutical quality of these preparations of A. afra. To identify and determine the levels of luteolin in the plant preparations, a validated HPLC assay was developed. Finally, the in situ perfused rat intestine model was used to determine the in vitro bioavailability, i.e. gastrointestinal permeability, of luteolin from solutions containing luteolin in pure form, FDAE, alginate-FDAE and PMMA-alginate-FDAE. The A. afra forms were obtained in moderate to good yields and FDAE was brown and hygroscopic in nature, the alginate beads dark brown free flowing and spherical in shape and the PMMA-alginate beads light brown in colour with rough edges. The A. afra plant forms on average contained 0.185 ± 0.24, 0.067 ± 0.014, 0.012 ± 0.071 μg/mg of free luteolin (n=3) in FDAE, alginate-FDAE and PMMA-alginate-FDAE respectively and 0.235 ± 0.026, 0.079 ± 0.093, 0.058 ± 0.082 μg/mg of total luteolin (n=3) in FDAE, alginate-FDAE and PMMAalginate- FDAE respectively. The Plumen values for intestinal uptake of luteolin were significantly higher from solutions of A. afra forms than the pure luteolin solution (i.e. Plumen values in the range of 0.02 - 0.035 cm/s for all plant forms vs Plumen values in the range of 0.010 - 0.014 cm/s for pure luteolin, t-test p = 0.0252). The permeability of luteolin in FDAE appeared to be slighter greater than that of the modified forms (Plumen values >0.03 cm/s for FDAE and Plumen values <0.03 cm/s for both modified forms). In summary, the results showed that, the modified A. afra forms; alginate-FDAE and PMMAalginate- FDAE were of acceptable pharmaceutical quality with luteolin better taken up in the plant forms than in its pure form. The A. afra forms prepared had similar rates of uptake (permeability) of free and total luteolin with the rates being highest for the FDAE. Collectively, these results indicate that alginate-FDAE and PMMA-alginate-FDAE bead forms should be suitable for use in a solid dosage form (e.g. tablet or capsule) of A. afra.

Malaria is one of the deadliest parasitic diseases worldwide, causing 219 million infections and 435 thousand deaths per year. As such, this mosquito-borne illness is a major target for global eradication efforts. One critical arm of the eradication strategy is chemotherapy. For a therapeutic to advance the eradication agenda, it must cure the patient of infection and eliminate transmission stage parasites (called gametocytes) from the blood, thereby breaking the cycle of transmission. Currently, first-line treatments against malaria infection consist of an artemisinin derivative in combination with another antimalarial drug from a different drug class. Although artemisinin and its derivatives are highly efficacious at curing malaria, these drugs are ineffective at preventing disease transmission. However, recent in vivo studies have suggested that whole plant Artemisia annua (the botanical source of artemisinin) delivered as tea can cure patients of infection and eliminate transmission stage parasites from the bloodstream. To validate these in vivo results in vitro, experiments were performed to measure the killing efficacy of A. annua and A. afra tea infusions against three different stages of the parasite life cycle— one stage of the asexual cycle, immature gametocytes, and mature gametocytes. Killing effects were observed using light microscopy and gametocyte gene-specific RT-qPCR analyses. Results suggested that A. annua tea was nearly as effective as artemisinin at killing all three tested stages of the parasite. A. afra tea, which contains low levels of artemisinin, showed comparable killing efficacy against late stage gametocytes, but not against the other two tested stages. These results supported the notion that A. annua tea is an effective antimalarial and also provides evidence that both A. annua and A. afra teas may be a viable therapeutic option for eliminating gametocytes during human infection.

Thesis (M.A. Agricultural Management (Plant Production)) -- University of Limpopo, 2022 / Artemisia afra Jacq. ex Willd is a common medicinal plant in Africa belonging to the Asteraceae plant family. It has been used for decades as a treatment for cold related illnesses and a variety of other ailments such as asthma, malaria and kidney disorders. As such this has led to its high demand and it is faced with threats of overharvesting. As the plant is naturally occurring and not widely cultivated, it’s seed biology and germination properties are not documented. The optimum requirements (temperature and photoperiod) for seed germination as well as the effects of Moringa based biostimulant and different scarification methods were investigated on A. afra seeds in this study. The seeds were exposed to different scarification methods including physical scarification (rubbing using sandpaper), hot water soaking, cold water soaking, acid treatment (H2SO4) and fermentation (using effective microorganisms) while the untreated seeds served as the control under all conditions. Moringa based biostimulant was also used as a seed priming agent at 0, 0.5, 1 and 3% levels. The seeds were then placed under a variety of photoperiods including constant light, alternating light (16/8 hours) and continuous darkness at different temperatures (15°C, 25°C and 35°C). Germination percentages and mean germination time (MGT) were calculated from the results and Statistix 10.0 software was used at the probability level of 5%, to compare the mean values. It was observed that under the different temperatures without any other treatments, the highest germination rate (70%) was achieved at a 25°C. No germination (0%) was obtained at both 15°C and 35°C in all the experiments including scarification methods and photoperiods. Under the scarification treatments, soaking in hot and cold water scarification improved germination with final germination of 70%, compared to the other scarification methods (Physical at 20%, acid treatment at 20% and control at 10%). There was no germination observed (0%) for the fermented seeds in all the experiments. On the photoperiod treatments, the highest germination percentage of 70% was observed under alternating light condition. However, when mean germination time was calculated, continuous xiv darkness resulted in shortest MGT of 11 days compared to 13 days for both alternating light and for continuous light in this set of experiment. Seed priming with Moringa based biostimulant under alternating light conditions and at 25°C resulted in improved MGT. Under the investigated treatments, the use of 3% biostimulant exerted the highest improvement on germination with germination percentage of 62% and the shortest MGT of 7 days compared to the other treatments. Generally the final germination (%) and time of A. afra seeds were affected by scarification, temperature, light and priming using Moringa based biostimulant. The information generated from this study is important because it will contribute in bridging the scientific gap on the information that has never been documented on optimum requirements for effective propagation of A. afra through seeds. As such this information will be useful for small scale farmers and medicinal plant growers who are willing to recover the lost populations of A. afra through seed propagation thus improving their supply and income / National Research Foundation (NRF)

Magister Pharmaceuticae - MPharm / Leonotis leonurus (L. leonurus) and Artemisia afra (A. afra) are two of the most commonly used medicinal plants in South Africa traditionally advocated for use in asthma. However, proper scientific studies to validate these claimed uses are lacking and little is known about the mechanisms for this effect. These plants contain flavonoids, which are reported to have smooth muscle relaxant activity and may be responsible for the activity of these two plants. The objectives of this study were to: (1) determine and compare the flavonoid profiles and levels in A. afra and L. leonurus, (2) compare the respiratory smooth muscle relaxant effects of freeze-dried aqueous extracts of A. afra and L. leonurus and (3) investigate whether K⁺ - channel activation (i.e. KATP channel) is one possible mechanism of action that can explain the effect obtained in traditional use of these two plants. It was hypothesized that: (1) the flavonoid levels and profile of A. afra would be greater than the flavonoid levels and profile of L. leonurus, (2) A. afra would have a more potent respiratory muscle relaxant effect than L. leonurus and (3) A. afra and L. leonurus will inhibit K⁺ - induced contractions in a superior manner than carbachol and histamine - induced contractions. To realize these objectives, freeze-dried aqueous extracts (FDAE) of the dried leaves of the two plants were prepared. A validated HPLC assay was developed and used to identify and determine the levels of luteolin in the plant preparations. Solutions of the plant extracts were studied in the isolated guinea-pig trachea tissue preparation in the presence of carbachol, histamine and KCL. The possible mechanism of action of the two plants was determined by cumulative log dose-response curves (LDRC) for carbachol, histamine and KCL in the absence and presence of 1, 30 and 100 mg/ml solutions of the plant extracts. The flavonoid profile of un-hydrolyzed and hydrolyzed L. leonurus was greater than that of un-hydrolyzed and hydrolyzed A. afra. The levels of free and total luteolin in A. afra FDAE (8.977 ± 0.73 μg/ml and 16.394 ± 0.884 μg/ml, respectively) were significantly (p < 0.001) higher than that in L. leonurus FDAE (0.929 ± 0.066 μg/ml and 3.093 ± 0.531 μg/ml, respectively). L. leonurus and A. afra relaxed tracheal smooth muscles contracted with histamine, KCL and carbachol in a dose dependent manner. The degree of relaxant activity of L. leonurus versus the three inducers of contraction (agonists) could be classified as KCL > carbachol > histamine, with EC₅₀ values of 9.87, 29.34 and 94.76 mg/ml, respectively. The A. afra tracheal smooth muscle relaxant activity was categorized as carbachol > histamine > KCL, with EC₅₀ values of 13.93, 15.47 and 19.88 mg/ml, respectively. Overall, A. afra which contained the higher levels of luteolin, was more potent at relaxing the guinea pig tracheal smooth muscle than L. leonurus. Collectively, the results confirm that aqueous solutions of A. afra and L. leonurus as used in local traditional practice have potent but different degrees of bronchodilator activities that could be useful in the treatment of asthma, and that these actions may be related to each plant's luteolin (or flavonoid) levels. Moreover it is very unlikely that KATP channels are primarily responsible for the actions of A. afra and L. leonurus, but rather that more than one mechanism of action is involved in the tracheal smooth muscle relaxant effects of these two plants. / National Research Foundation

>Magister Scientiae - MSc / Current research classifies Type 2 diabetes as most prevalent non-communicable diseases in South Africa. Approximately 285 million people are affected globally with an expected increase to 595 million by the year 2035. Synthetic first-line drugs in the treatment of Type 2 diabetes, have been shown to have an efficacy rate of approximately 43% as a result of poor drug uptake and metabolism. Furthermore, given South Africa’s uniquely diverse botanical heritage, herbs commonly used traditional medicine have shown promise in the treatment of Type 2 diabetes.

Thesis (MTech (Horticulture))--Cape Peninsula University of Technology, 2013. / This study evaluated the effects of supplementary phosphorus, copper and zinc alone and in conjunction with arbuscular mycorrhiza on Artemisia afra grown in a simulated soil medium. The treatments consisted two groups. Group A had no mycorrhizal inoculation and 1) no supplementary fertilization, 2) supplementary zinc application, 3) supplementary copper 4) supplementary phosphorus 5) supplementary zinc and copper, 6) supplementary zinc and phosphorus, 7) supplementary copper and phosphorus, 8) supplementary zinc, copper, and phosphorus. Group B had mycorrhizal inoculation in combination with 9) no supplementary fertilization, 10) supplementary zinc application, 11) supplementary copper 12) supplementary phosphorus 13) supplementary zinc and copper, 14) supplementary zinc and phosphorus, 15) supplementary copper and phosphorus, 16) supplementary zinc, copper, and phosphorus. There was also a pilot study into the pH range most suitable for the cultivation of A. afra in a hydroponic system, which was used to refine the mycorrhizal investigation, as pH has influences with regards to nutrient uptake of plants.

The aim of this study was to investigate the effect the plant matrix and the structure of the flavonoid (i.e. whether aglycone or glycoside) may have on the gastrointestinal uptake and metabolism of luteolin derivatives from Artemisia afra traditional plant medicine. Specifically, how these two factors influenced the intestinal uptake and disposition of luteolin derivatives in pure and in Artemisia afra plant extract forms were to be assessed by investigating the uptake and metabolism of the luteolin derivatives in human intestinal epithelial Caco-2 cells and the perfused rat intestinal loop. To realize this aim, the following were determined: (1) identification and characterization of major luteolin derivatives found in Artemisia afra, (2) the effect of the plant matrix on the uptake of luteolin derivatives in Artemisia afra aqueous-extract forms across the Caco-2 cell monolayer, (3) the effect of the plant matrix on the absorption and metabolism of luteolin derivatives in Artemisia afra aqueous-extract forms in the perfused rat small intestine, (4) the effect of gut contents on the uptake and metabolism of luteolin derivatives in intestinal loop and (5) the metabolic profiles of luteolin derivatives obtained for the pure solutions versus plant aqueous extract solutions in Caco-2 cells and the rat intestine. / Philosophiae Doctor - PhD