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

Doctor Pharmaceuticae - DPharm / The central nervous system (CNS), consisting of the brain and the spinal cord, is responsible for integrating sensory information and influencing most bodily functions . The CNS is protected from toxic and pathogenic agents in the blood by permeability barrier mechanisms. These barrier mechanisms, specifically the blood brain barrier (BBB) presents a challenge for the discovery of CNS active drugs as it is requirement for these drugs to permeate the BBB to reach their target site in the CNS. The conventional processes of drug design and discovery from natural products are time consuming, tedious, expensive and have a high failure rate. It has been reported from various studies that the use of computational modelling and simulations in drug design and discovery is less costly and less time-consuming with a greater chance of success than the conventional processes. The process of drug discovery and design can, therefore, be easily carried out using proven computer models, software, and web-based tools . / 2023

This thesis discussed the cardiovascular effects of the aqueous leaf extract and a fraction of the methanol extract of Leonotis leonurus, a plant commonly used in traditional medicine in South Africa for the treatment of hypertension and other cardiac problems. The cardiovascular effects was tested on anaesthetized normotensive male Wistar rats and isolated perfused rat hearts.

This thesis discussed the cardiovascular effects of the aqueous leaf extract and a fraction of the methanol extract of Leonotis leonurus, a plant commonly used in traditional medicine in South Africa for the treatment of hypertension and other cardiac problems. The cardiovascular effects was tested on anaesthetized normotensive male Wistar rats and isolated perfused rat hearts.

Magister Pharmaceuticae - MPharm / This thesis discussed the cardiovascular effects of the aqueous leaf extract and a fraction of the methanol extract of Leonotis leonurus, a plant commonly used in traditional medicine in South Africa for the treatment of hypertension and other cardiac problems. The cardiovascular effects was tested on anaesthetized normotensive male Wistar rats and isolated perfused rat hearts. / South Africa

The rising costs of prescription drugs in the maintenance of personal health and wellbeing have increased the interest in medicinal plants. The World Health Organization estimates that 65 percent-80 percent of the world’s population use traditional medicine as their primary form of health care. In this project the focus has been on the use of Leonotis leonurus extracts as a traditional medicine. The major chemical constituent of this plant is marrubiin, which is a diterpenoid labdane lactone formed from a precursor called premarrubiin. Aqueous and acetone extract (AL and OL extract, respectively) of this plant has been found to have an antithrombotic effect, with IC50 values of 3mg/ml and 6mg/ml, respectively. The extracts also have an effect on fibrinolysis, where the lysis time was decreased by more than 50 percent by the organic extract and standard marrubiin. In whole blood ADP-induced platelet aggregation, the organic extract inhibited aggregation by 68 percent at a final concentration of 138μg/ml (equivalent to 7.2μg/ml marrubiin). Marrubiin has also been screened for antithrombotic/anticoagulant activity; no antithrombotic activity has been observed but it increased the rate of fibrinolysis, by decreasing lysis time by 64 percent and also decreasing fibrin formation. From these findings it can be concluded that marrubiin has a fibrinolytic effect and antiplatelet aggregation effect. In the diabetic studies, in hyperglycemic condition, the OL (10μg/ml) extract and standard marrubiin significantly increased insulin secretion by 200 percent (2-fold) and 400 percent (4-fold), respectively, with respect to the control. The OL extract and standard marrubiin stimulated the release of insulin, the stimulatory index was significantly increased by 450 percent (4.5-fold) and 500 percent (5-fold), respectively, with respect to the control. In the apoptotic studies, in the normoglycemic and hyperglycemic conditions, the OL extract decreased the occurrence of apoptosis, in a dose-dependent manner, with the lower concentrations inducing apoptosis significantly higher than the relevant controls. Standard marrubiin did not have an effect on apoptosis in hyperglycemic condition, but it decreased the occurrence of apoptosis by 200 percent (2-fold) under normoglycemic conditions. The OL extract increased proliferation by 148 percent (1.48- fold) and 155 percent (1.55-fold) in normoglycemic and hyperglycemic conditions, respectively. The same effect was observed for standard marrubiin, where, proliferation was increased by 180 percent (1.8-fold) and 200 percent (2.0-fold) in normoglycemic and hyperglycemic conditions, respectively. RT-PCR displayed that standard marrubiin inhibited the expression of insulin by 50 percent under normoglycemic conditions.

Philosophiae Doctor - PhD / Plants used in traditional medicines have served as sources of some of the drug compounds used in medicines today, and could still serve as leads for then development of new drugs to treat existing chronic diseases such as hypertension. This study was aimed at the isolation and identification of a cardio-active compound from L. leonurus, a plant commonly used in traditional medicines in South Africa for the treatment of hypertension and other cardiac problems. The possible mechanisms by which the isolated compound produced its effect on the cardiovascular system were explored using the anaesthetized normotensive rat model.Fractionation of the organic extracts of the leaves led to the isolation of a novel diterpene,(13S)-9 , 13 -epoxylabda-6 (19),15(14)diol dilactone (EDD) whose structure was elucidated using infra red (IR), nuclear magnetic resonance (NMR), mass spectroscopy(MS), and X-ray diffraction analysis. In anaesthetized normotensive male Wistar rats, EDD(0.5 mg/kg – 5.0 mg/kg; IV) produced slight non-significant decreases in systolic pressure(SP), diastolic pressure (DP), and mean arterial pressure (MAP) with the lower (0.5 mg/kg– 2.0 mg/kg) doses, while significant increases in SP, DP and MAP occurred with the higher (3.0 mg/kg – 5.0 mg/kg) doses. All doses of EDD administered also produced significant decreases in heart rate (HR).Prazosin and reserpine pre-treatment abolished the vasoconstrictive effect of EDD,suggesting an indirect vasoconstrictive effect for EDD via the release of catecholamines.Atenolol pre-treatment led to increases in the negative chronotropic effect of EDD, while the positive chronotropic effect of dobutamine was significantly decreased by EDD,suggesting the involvement of the 1 adrenoceptor in the negative chronotropic effect of EDD. In animals pre-treated with verapamil, a cardio-selective Ca2+ channel blocker, no significant changes in HR occurred with all EDD doses, but HR values were significantly lower than those obtained with EDD in non pre-treated animals.The results of this study indicate that (13S)-9 , 13 -epoxylabda-6 (19),15(14)diol dilactone, a novel dilactone diterpene isolated from the leaves of L. leonurus has an effect on the cardiovascular system. EDD exhibits a dual effect on the cardiovascular system by producing a vasoconstrictive effect accompanied by bradycardia. The vasoconstrictive effect of EDD is probably due to the release of catecholamines, while the negative chronotropic effect is probably due to 1 adrenoceptor antagonism. Further studies are however required to fully determine the mechanism by which EDD produces its cardiovascular effects.

Commercial marrubiin, aqueous and organic extracts of Leonotis leonurus were tested in vitro for their anticoagulant and antiplatelet activities. The aqueous extract inhibited platelet aggregation by 69.5 percent (100 μg/mL), while the organic extract (100 μg/mL) and marrubiin (5 μg/mL) showed 92.5 percent and 91.6 percent inhibition, respectively, by inhibiting the binding of fibrinogen to glycoprotein IIb/IIIa receptor in a concentration dependent manner. The extracts significantly prolonged activated partial thromboplastin time compared to untreated plasma controls. Fibrin and D-Dimer formation were drastically decreased. The extracts and marrubiin concentration-dependently inhibited calcium mobilization induced by collagen and thrombin. The formation of thromboxane A2 was also significantly reduced by both the extracts and marrubiin. Protein secretion and platelet adhesion were significantly reduced by both the extracts and marrubiin. The organic extract and marrubiin showed a more pronounced effect than the aqueous extracts in all the in vitro assays. The ex-vivo animal model confirmed the results obtained in vitro. Similar to the in vitro studies, activated partial thromboplastin time clotting time was prolonged by marrubiin and the number of aggregated platelets were significantly reduced relative to aspirin. The findings reflect that marrubiin largely contributes to the organic extract's anticoagulant and antiplatelet effect in vitro. INS-1 cells were cultured under normo- and hyperglycaemic conditions. Marrubiin and the two Leonotis leonurus extracts were screened for anti-diabetic activity in vitro. The stimulatory index of INS-1 cells cultured under hyperglycaemic conditions was significantly increased by 60 percent and 61 percent (p<0.01; n=5) in cells exposed to the organic extract (10 μg/mL) and marrubiin (500 ng/mL), respectively, relative to the normoglycaemic conditions. The gene expression of insulin was significantly increased by 76.5 and 71 percent, and of glucose transporter-2 by 93 and 92.5 percent for marrubiin and the organic extract, respectively, under the same conditions stipulated above (p<0.01; n=4). The extract and marrubiin similarly showed an increase in respiratory rate under hyperglycaemic conditions. Marrubiin increased insulin secretion, HDL-cholesterol, while it decreased total cholesterol, LDL-cholesterol and the atherogenic index in the in vivo rat model.

Doctor Pharmaceuticae - DPharm / An aqueous extract prepared from the leaves and smaller stems of Leonotis leonurus was used to investigate the potential effects on certain cardiovascular parameters, such as left ventricular systolic pressure, end-diastolic pressure, developed pressure, heart rate, cardiac work and coronary perfusion pressure in isolated rat hearts. Hearts were perfused at constant flow for 3min using the modified Langendorf! perfused model of the heart. Effects of adrenaline and digoxin solutions on the isolated heart were compared to that of the plant extract. Adrenaline produced both positive inotropic and chronotropic effects. Adrenaline increased (p<O.Ol) the left ventricular systolic pressure and hence the left ventricular developed pressure by 40.6% and 43.9% at peak, and 24.3% and 31.9%, after 3min, respectively. Simultaneously, the heart rate and the cardiac work were increased (p<0.01) by 22.5% and 89.4% at peak, and 24.6% and 63%, after 3rnin, respectively. There were no significant effects on the left ventricular diastolic pressure and the coronary perfusion pressure. Digoxin solution (2.5ng/ml) significantly (p<O.Ol) increased the left ventricular systolic pressure by 5.1% after 3min and the left ventricular diastolic pressure by 9.7% at peak and 5.3% after 3min. The heart rate was significantly (p<O.OI) decreased by 3.7% at peak. The cardiac work was increased by 4.5% after 3rnin. Digoxin did not significantly affect the left end diastolic pressure and the coronary perfusion pressure. The extract of Leonons leonurus at O.lmg/ml increased (p<O.OI) the left ventricular systolic pressure and hence the left ventricular diastolic pressure by 9.7% and 10.7% at peak, and 5.4% and 5.5% after 3rnin, respectively. The cardiac work was increased (p<O.Ol) by 10.1% at peak. Leonotis leonurus (0.1mg/ml) did not significantly affect the left ventricular end diastolic pressure, the heart rate and the coronary perfusion pressure. At 0.5mg/ml, the left ventricular systolic pressure and hence the left ventricular diastolic pressure were increased (p<0.01) by 14.8% and 15.4% at peak and 7.4% and 7.8% after 3rnin, respectively with a corresponding decrease (p<O.OI) in the coronary perfusion pressure of 8.5% at peak and 4.4% after 3rnin. The cardiac work was increased (p<O.OI) by 13.6% at peak and 5.2% after 3rnin. The extract at 1.0mg/ml increased (p<O.Ol) the left ventricular systolic pressure and hence the left ventricular diastolic pressure by 25.4% and 29.4% at Peak, and 23.1% and 26.3% after 3rnin, respectively. The heart rate was reduced (p<O.OI) by 34.7% at peak and 28.3% after 3min. The cardiac work and the coronary perfusion pressure were decreased (p<O.OI) by 15.9% and 12.1% at Peak and 3.3% and 11.4% after 3rnin. However, at 2.0mg/ml, the left ventricular systolic pressure and the left ventricular diastolic pressure were increased (p<O.OI) by 14.9% at peak. The left ventricular diastolic pressure was decreased (p<O.OI)by 9.8% over the 3rnin. The heart rate was drastically decreased (p<O.OI) by 42.7% after 3rnin. The cardiac work was reduced (p<O.Ol) by 48.8% over the 3min period. Also, the coronary perfusion pressure was decreased (p<0.01) by 16.9% at peak. Thus, Leonatis leonurus produced both positive inotropic and negative chronotropic effects after 3min perfusion, accompanied by a decreased coronary perfusion pressure. Thus, it appears that the extract seemed to contain certain constituents associated with positive inotropic and negative chronotropic agents as wel! as constituents associated with coronary vasodilation. However, at the higher concentration, it seemed to contain some constituents associated with toxic effects on the isolated heart. Therefore, further studies are needed to isolate the various constituents and examine their possible pharmacological effects on the heart individually before it could be considered safe to recommend this plant for its use in the treatment of cardiovascular disease.