Magister Pharmaceuticae - MPharm / Leonotis leonurus, a South African indigenous medicinal plant, is frequently used in the form of a tea. However, this dosage form has many disadvantages. Consequently three L. leonurus solid extract preparations were prepared and explored as possible replacements of the tea form, but very little was known about their physical and chemical stability during storage. The specific objectives were to: (i) prepare a freeze dried aqueous extract (FDAE), 20 % aqueous ethanol (Aq EtOH) extract and calcium alginate beads of the FDAE form of L. leonurus, (ii) characterize the extracts using parameters of select physical and chemical features and, (iii) determine the long-term stability of the extracts. It was hypothesised that the Aq EtOH extract would contain higher levels of chemical marker compounds (marrubiin and leonurine) than the FDAE and calcium alginate FDAE beads of L. leonurus and, that the calcium alginate FDAE beads would have greater stability (i.e. longer shelf-life) than the FDAE and the Aq EtOH extract. The three L. leonurus solid extracts were prepared using accepted published methods. For the physical characterization of the extracts, the organoleptic properties were determined using the natural senses (e.g. sight, smell, taste, etc.) and for chemical characterization, total phenol content (TPC; using the Folin-Ciocalteu reagent method), total flavonoid content (TFC; using aluminium chloride-methanol solution) and antioxidant activity (using the -diphenyl-2-picryl-hydrazyl (DPPH) assay). To establish the long-term stability of the preparations, encapsulated L. leonurus solid extracts was stored in sealed standard plastic containers at four conditions: (A), room temperature of 24 ˚C ± 5 ˚C; (B), fixed temperature of 30˚C ± 5 ˚C and (C), elevated temperature of 40˚C ± 5 ˚C for 6 months, and (D), accelerated stability test conditions of 40˚C ± 5 ˚C / 75 % RH for 4 weeks. Samples of the stored encapsulated preparations were collected periodically and assessed for changes in organoleptic properties, TPC, TFC, antioxidant activity levels and marker compound (i.e. marrubiin and leonurine) levels. The latter was determined by validated HPLC assay. Yields of 19.9, 12.82 and 10.7 % of FDAE, Aq EtOH extract and calcium alginate FDAE beads were obtained, respectively. Physically the calcium alginate beads contained less moisture (1.86 %) than the FDAE (3.77 %) and Aq EtOH (2.91 %). Chemically the FDAE, Aq EtOH extract and calcium alginate FDAE beads respectively had appreciable and similar TPC (i.e.7.86, 7.52 &, 6.94 mg GAE/g; p > 0.05; Anova) and TFC (i.e. 4.30, 4.47 & 3.67 mg QE/g; p > 0.05; Anova) levels, but variable amounts of marrubiin (i.e. 22.5, 17.5, and 0.4 ug/mg plant extract) and leonurine (i.e. 2.0, 1.4 and 0.7 ug/mg plant extract), respectively. The antioxidant activity levels were also different i.e. EC50 values of 7.71, 6.66 and 11.53 mg/mL (student t-test p-value of < 0.0001; ANOVA-test; p< 0.05) for the FDAE, Aq EtOH extract and calcium alginate FDAE beads, respectively. During storage (i.e. stability study) the L. leonurus solid extracts generally remained physically unaffected by temperature (i.e. no significant change in organoleptic features), but when exposed to humidity the FDAE and Aq EtOH extracts showed clear signs of physical degradation i.e. changed from being flaky powders to sticky melted masses, while the calcium alginate beads remained unchanged. Within 1 month storage at RT, 30 °C, 40 °C and 1 week at 40 °C / 75 % RH the TPC of the encapsulated FDAE decreased significantly by 61, 60, 58 and 52 %, respectively, that for the encapsulated Aq EtOH extract by 61, 54, 46 and 50 %, respectively, and for calcium alginate FDAE beads by 66, 71, 59 and 57 %, respectively. Using TPC as a stability parameter all three encapsulated extracts had very short shelf-lives ranging from 1.24 weeks (0.31 months) to 3.72 weeks (0.93 months). Under the same conditions and storage periods (i.e. 1 month & 1 week) the TFC of the encapsulated FDAE decreased significantly by 25, 25, 29 and 66 %, respectively, for encapsulated Aq EtOH extract by 26, 26, 23 and 70 %, respectively, and the calcium alginate FDAE beads by 55, 55, 52 and 64 %, respectively. The results obtained for TFC was thus similar to that obtained for the TPC data. Based on the TFC data all three encapsulated extracts had very short shelf-lives ranging, from 1.56 weeks (0.39 months) to 6.76 weeks (1.69 months). Under the same conditions and storage periods (i.e. 1 month & 1 week) as that used to determine TPC and TFC, the antioxidant activity of the extracts changed little, i.e. decreased by 0.2, 0.1, 0.8 and 2 %, respectively for FDAE, by 0.7 %, 1 %, 0.1 % and 5.3 %, respectively for the Aq EtOH and by 2, 2, 1.4 and 0.8 %, respectively for the calcium alginate FDAE beads. Moreover, based on antioxidant activity, all three encapsulated extracts had relatively long shelf-lives ranging from 15.6 weeks (3.9 months) to 22.4 weeks (5.6 months). Finally, the determination of the stability of the encapsulated L. leonurus extracts stored under stress conditions (i.e. 40 °C / 75 % RH) and based on marker compound levels was unresolved. Between the time of extract preparation and characterisation until start of the stability study the marrubiin levels in the FDAE, Aq. ETOH and calcium beads had decreased from 22.5, 17.5, and 0.4 ug/mg plant extract, respectively, to 0.30, 0.11, 0.30 μg/mg, respectively, and the leonurine levels from 2.0, 1.4 and 0.7 to 0.46, 0.38 and 0.09 μg/mg, respectively and was too low to conduct a meaningful stability study with the developed validated assay. Overall, all three the encapsulated L. leonurus solid extracts studied were clearly very unstable and did not have suitable long-term storage stability. The modification of the freeze-dried aqueous extract of L. leonurus into a calcium alginate bead form seemed to combat physical instability but did not improve the chemical instability of the aqueous extract. It is therefore recommended that the addition of excipients or other post extract modification (e.g. production of phytosomes) be explored to combat the hygroscopicity of L. leonurus FDAE and ultimately improve its overall product stability.

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 Pharmaceuticae - Mpharm / The aim of this study was to determine quality control specifications needed for a dossier and an investigator's brochure of A. afra capsules, which can be used to motivate the registration and clinical testing of A. afra capsules in chronic asthma. The specific objectives were: (1) to establish the minimum product quality requirements for registration of A. afra capsules, (2) to prepare and pharmaceutically characterize a capsule product of A. afra freeze dried aqueous extract (FDAE) suitable for registration, and (3) to identify pharmaceutical product quality aspects of an investigator's brochure (IB) that would be appropriate for use in motivating a clinical trial of A. afra capsules in chronic asthma.