Diabetes mellitus (DM) represents a series of metabolic conditions associated with hyperglycemia and caused by defects in insulin secretion, and/ insulin action. Exposure to chronic hyperglycemia may result in microvascular complications in the retina, kidney or peripheral nerves. According to the World Health Organization (WHO) global burden of disease, more than 176 million people are diabetic with about two thirds of these living in developing countries. With a long course and serious complications that often result in high incidences of mobility and mortality rate, the treatment of diabetes is often costly. The management of this disease is not without side effects and this is a challenge to the medical system. This has led the researches to seek new antidiabetic agents from plants. Acetone extract of 8 plants namely Terminalia sericea Burch. Ex DC, Euclea natalensis A.DC, Warbugia salutaris Bertol.f.) Chiov., Artemisia afra Jacq.ex Willd., Aloe ferox Mill, Sclerocarya birrea (A.Richi.) Hochst. subsp. caffra , Spirostachys Africana Sond and Psidium guajava L were evaluated for antidiabetic and antioxidant properties. In addition extracts were tested for cytotoxicity. Different parts of all these plants are traditionally used in South Africa for diabetes treatment. Plants were selected based on ethnobotanical information and phytochemical constituents. For determining inhibitory activity against each enzyme (α-glucosidase and α- amylase), all extracts were tested at concentration that ranged from 2x10-5 to 0.2mg/ml for α-glucosidase and 0.025 to 1.25mg/ml for α-amylase and fifty percent inhibition or higher was taken as significant (p<0.05). The extracts of A. ferox and S. africana showed no inhibition against α-glucosidase at the highest concentration tested (0.2mg/ml) whereas A. afra showed weak inhibition (47.15%). T. sericea showed to be a potent inhibitor of α-glucosidase exhibiting 97.44 % inhibition of the enzyme (p<0.05). W. salutaris, S birrea and E. natalensis also showed good activity on α-glucosidase as they demonstrated 71.84; 97.44 and 92.60 % inhibition respectively (p<0.05). Other plant extracts such as A. ferox and S. africana did not exhibit any activity on α-glucosidase. T. sericea and S. birrea showed the best inhibitory activity on α-amylase enzyme, exhibiting 91.91 and 94.94 % inhibition respectively at 1.25mg/ml. A. afra, E. natalensis, P. guajava and W. salutaris also showed good inhibitory activity on -amylase enzyme at 1.25mg/ml which was the highest concentration tested (p<0.05). Low levels of plasma antioxidants is a risk factor associated with diabetes therefore, it has been suggested that plant-based medicines that contain antioxidant properties add an advantage in curbing complications that arise during DM aetiology. The antioxidant activity of plant extracts was carried out using 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) assay. Six plant extracts which showed good α-glucosidase and α-amylase inhibitory activity were evaluated for antioxidant activity. The radical scavenging activity was measured in terms of the amount of antioxidants necessary to decrease the initial DPPH absorbance (EC50). The EC50 is the amount of antioxidants necessary to decrease initial DPPH absorbance by 50%. All 6 tested plant extracts showed good activity. W. salutaris and T. sericea demonstrated the highest activity exhibiting EC50 values of 5.08 and 5.56βg/ml respectively as compared to ascorbic acid/Vitamin C (EC50=2.52μg/ml), a well- known potent antioxidant. This was followed by P. guajava (EC50=6.97μg/ml); E. natalensis (EC50=8.46μg/ml) and S. birrea (EC50=9.41μg/ml). A. ferox showed EC50 value of 48.53μg/ml. It has been suggested that plant extracts and compounds must undergo toxicity test for safety before drug discovery is taken into consideration. Due to the large number of plants screened in this study and limited resources in our laboratory, only the acetone extract of T. sericea (which demonstrated good α-glucosidase and -amylase inhibitory activities) was tested for cytotoxicity. Acetone extract of T. sericea demonstrated moderate toxicity against primary vervet monkey kidney cells (VK) cells exhibiting IC50 values of 20.94 μg/ml when tested at 400μg/ml. Consequently, the acetone extract of T. sericea was selected for the isolation and identification of bioactive compounds. A bio-assay guided fractionation of the acetone extract of T. sericea led to the isolation of 4 pure compounds namely β-sitosterol, β-sitosterol-3-acetate, lupeol and 3-onestigmasterol and two sets of mixtures of isomers (epicatechin-catechin; MI1 and epigallocatechingallocatechin; MI2). Antidiabetic, antioxidant and cytotoxicity activities of isolated compounds were evaluated. μ–Sitosterol and lupeol showed best inhibitory activity on α-glucosidase exhibiting 50% inhibitory concentration (IC50) value of 54.50 μM and 66.48 μM respectively (p<0.05). This was followed by the MI2; epigallocatechin-gallocatechin (IC50=119.34 μM); β-sitosterol-3-acetate (IC50=129.34 μM); 3-one-stigmasterol (IC50=164.87 μM) and the MI1; epicatechin-catechin (IC50=255.76 μM). During the evaluation of purified compound’s inhibitory activity on α-amylase, compounds of interest were lupeol and β-sitosterol which exhibited IC50 values of 140.72 μM and 216.02 μM respectively as compared to the positive drug-control acarbose (IC50=65.25 μM). Epicatechincatechin and epigallocatechin-gallocatechin also demonstrated α-amylase inhibitory properties and the IC50 values were found to be lower than 100μg/ml. Epigallocatechin-gallocatechin, epicatechin-catechin and lupeol showed good free radical scavenging activity as they inhibited DPPH by 98.19; 96.98 and 70.90 % at 100ìg/ml respectively (p<0.05). The DPPH scavenging activity was very low in case of 3-one-stigmasterol (21.5% inhibition), whilst β-sitosterol and its derivative β-sitosterol-3-acetate did not show any activity. During cytotoxicity evaluation of pure compounds against monkey kidney cells, all the compounds except β-sitosterol did not inhibit the growth of these cells lines at the highest concentration tested (200μg/ml). β-Sitosterol showed moderate toxicity exhibiting IC50 values of 197.72 μM. β- Sitosterol-3-acetate, epicatechin-catechin, lupeol and epigallocatechin-gallocatechin were found to be non-toxic to Vero cells as 100% cell viability was observed when Vero cells were exposed to these samples at 200μg/ml. The compounds isolated and the extract of T. sericea demonstrated significant antidiabetic and antioxidant properties as compared to well known drugs acarbose (a known -glucosidase and α- amylase inhibitor) and Vitamin C (a well known antioxidant). This study is the first to report α- glucosidase, α-amylase and antioxidant properties of epicatechin-catechin, epigallocatechingallocatechin, β-sitosterol-3-acetate and stigma-4-ene-3-one isolated from T. sericea. In addition, epicatechin-catechin, epigallocatechin-gallocatechin, β-sitosterol-3-acetate and stigma-4-ene-3-one are isolated from T. sericea for the first time. Overall all results scientifically validated the traditional use of the bark of T. sericea for diabetes in South Africa. / Dissertation (MSc)--University of Pretoria, 2011. / Plant Science / unrestricted

South Africa is home to a wide diversity of cultural groups, all of which utilize the flora for a variety of purposes. This is true with regard to traditional medicine systems which are similar to those of the rest of Africa south of the Sahara, with diviners (sangomas) and herbalists (inyangas) as the key health providers. In addition, the Country is rich in plant diversity with some 30 000 species of flowering plants - almost one tenth of the worlds recorded higher plants. This incorporates a large diversity of plants including trees, shrubs, herbs, bulbs and corms. The adverse effects of traditional medicinal plants and natural products are not well documented in the literature. Recently, many plants used as food or in traditional medicine have been shown to be potentially mutagenic using in vitro assays. Thus, the scientific evaluation of traditional medicine and medicinal plants is very important to validate claims made on safety and efficiency of such usages. After a survey of the available ethnobotanical literature, ten trees used in South African traditional medicine were selected. These species were: Acacia niolotica subspecies kraussiana, Acacia sieberiana, Albizia adianthifolia, Combretum kraussii, Faidherbia albida, Ficus sur, Prunus africana, Salix mucronata, Terminalia sericea and Trichilia dregeana. Plant parts including leaf, root and bark were collected from each of the selected trees (exceptions were Albizia adianthifolia, Faidherbia albida, Terminalia sericea and Prunus africana) and extracted using ethyl acetate, ethanol and water individually to ensure the extraction of compounds over a wide range of polarities. The extracts (in total, 78) were screened for antibacterial, anti-inflammatory (COX-1 and COX-2) and antiacetylcholinesterase activities and investigated for their potential mutagenic effects using the Ames test. Antibacterial activity was detected using the disc-diffusion and microdilution assays. The extracts were tested against Gram-positive bacteria: Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus and Gram-negative bacteria: Escherichia coli and Klebsiella pneumoniae. Of the 78 different plant extracts 111 tested (final amount of plant material was 1 mg per disc), 84% showed activity against Gram-positive bacteria. From this percentage, 20% also showed activity against Gram-negative bacteria. The best inhibition was observed with ethyl acetate and ethanol root extracts of Terminalia sericea against both Gram-positive and Gram-negative bacteria. In the micro-dilution assay, 55% of the plant extracts showed minimum inhibitory concentration (MIC) values ~ 1.56 mg/ml against Gram-positive and/or Gram-negative bacteria. The ethyl acetate bark extract of Acacia sieberiana and the root and bark ethyl acetate extracts of Acacia nilotica inhibited bacterial growth of both Gram-positive and Gram-negative bacteria at concentrations ~ 0.8 mg/ml. The aqueous leaf extracts of Acacia sieberiana had a low MIC value (0.3 mg/ml) against Gram-negative Kleibsiella pneumoniae and the ethyl acetate extracts of the root inhibited growth of Escherichia coli with an MIC value of 0.1 mg/ml. However, these two extracts showed no activity in the disc-diffusion assay. The MIC values of the neomycin (control) were 0.8 I-Ig/ml and 3.1 I-Ig/ml against Kleibsiella pneumoniae and Escherichia coli respectively. In the anti-inflammatory test, 70% of the plant extracts from different plant parts (leaf, root, bark) of the tree investigated showed strong inhibition in both the CQX-1 and CQX-2 bioassays. The CQX-2 inhibitory effects of aqueous extracts were generally lower when compared to the organic solvent extracts. However, water extracts of Acacia nilotica was an exception (~ 90%). In the acetylcholinesterase inhibitory test, 21% of the plant extracts were active at concentrations ~ 1 mg/ml using the micro-plate assay. The lowest IC50 value was 0.04 mg/ml obtained with an ethanol bark extract of Combretum kraussii. The IC50 value of the galanthamine (positive control) was 2 I-IM. None of the investigated plants showed any potential mutagenic effects with Salmonella typhymurium strain TA 98 using the Ames test. Using bioassay-guided fractionation, anolignan B was isolated from the ethyl acetate root extract of Terminalia sericea. Antibacterial activity of anolignan B was determined using the microdilution assay. The compound possessed activity against both Gram-positive and Gram-negative bacteria. The lowest MIC value (3.8 IJg/ml) was observed with Staphylococcus aureus. MIC value of the neomycin was 1.5 IJg/ml. Anti-inflammatory activity of anolignan B was detected using the CQX-1 and CQX-2 bioasays. The compound showed strong inhibitory activity against CQX-1 and weaker activity against CQX-2. The ICso values were 1.5 mM and 7.5 mM with CQX-1 and CQX-2 respectively. The ICso values of indomethacin were 0.003 mM and 0.186 mM against CQX-1 and CQX-2 respectively. There were no potential mutagenic effects showen by anolignan B against Salmonella typhimurium strain TA 98 in the Ames test. Isolation of anolignan B from Terminalia species and the antibacterial and anti-inflammatory activities observed in this work have not been reported previously and could therefore be recorded as novel biological activities for this compound. These results also support the idea that the use of ethnobotanical data can provide a valuable short cut by indicating plants with specific uses which might likely be sources of biologically active chemicals. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

MSc (Botany) / Department of Botany / See the attached abstract below

PhD (Chemistry) / Department of Chemistry / Terminalia sericea Burch. ex. DC (Combretaceae) is one of the 50 most popular medicinal plants in Africa. The fruit, leaves, stems and roots are commonly used for the treatment of cough, skin infections, diabetes, diarrhoea, venereal diseases and tuberculosis. However, the roots are most commonly used in the preparation of traditional medicines. Pharmacological studies have revealed that the crude root extracts display antibacterial activity against several Gram-positive and Gram-negative bacteria. Anolignan b, termilignan b and arjunic acid are reported to be the major antibacterial constituents present in the roots. Other compounds isolated from the roots include resveratrol-3-rutinoside, sericic acid, sericoside and arjunglucoside I. Authorities worldwide, including the Medicines Control Council of South Africa, have begun to regulate herbal drugs sold in the form of commercial formulations. Quality control of herbal drugs is challenging, since the chemical profiles of the raw materials may vary, depending on the origin of the plant material and the way that it was handled and processed. The chemistry, in turn, impacts on the safety and efficacy of the plant material. To date, there are no available data on parameters that can be used to standardise the quality of T. sericea raw materials. The aim of this study was therefore to provide information on the variation of the chemical constituents that contribute to the biological effects of the roots of T. sericea and also establish its safety. Since the compounds previously isolated from the roots were not commercially available, isolation of the major constituents of the roots was undertaken to obtain analytical standards. A crude dichloromethane:methanol (1:1) extract was initially fractionated using silica gel column chromatography, where after, some of the fractions were further purified using silica gel and Sephadex LH-20 column chromatography. Final purification of the enriched fractions was achieved using preparative high performance liquid chromatography coupled with mass spectrometry (prep-HPLC-MS). The structures of these compounds were subsequently elucidated using one- and two- dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry and identified as sericic acid (340 g), sericoside (500 g), resveratrol-3-rutinoside (240 mg) and arjunglucoside I (74 mg). The chemical variation within the crude root extracts of samples (n = 42) from ten populations in the Limpopo Province of South Africa, was determined using ultra performance liquid chromatography-mass spectrometry (UPLC-MS). A method was developed for the simultaneous determination of sericic acid, resveratrol-3-rutinoside, sericoside and arjungluicoside I in the extracts using UPLC with photodiode array detection (PDA). The method was validated according to the guidelines of the International Council for Harmonisation (ICH). A regression coefficient (R2) of 0.998 was obtained for sericic acid, resveratrol-3- rutinoside and arjunglucoside I, while the R2 value for sericoside was 0.999, indicating a linear relationship between the concentration and the detector response. Satisfactory limits of detection for sericic acid (25.2 ng/mL), sericoside (11.6 ng/mL), resveratrol-3-rutinoside (23.3 ng/mL) and arjunglucoside I (8.81 ng/mL) were determined. Recoveries of 98 % and 80% were obtained for samples spiked with 12.5 μg/mL and 25 μg/mL of resveratrol-3-rutinoside, respectively, indicating that the method is accurate. The intra- and inter-day variation in resveratrol-3-rutinoside concentration, measured over three days, indicated excellent analytical precision, since all the relative standard deviations were below 0.70 %. The quantitative data revealed that sericic acid (1.59 to 8.45 mg/g), sericoside (2.07 to 20.17 mg/g), resveratrol-3-rutinoside (0.65 to 29.82 mg/g) and arjunglucoside I (0.86 to 8.44 mg/g dry weight) were the major constituents of the root samples, but their concentrations were highly variable. Chemometric analysis of the aligned UPLC-MS data was used to investigate similarities and differences in the chemical profiles of the samples using an untargeted approach. A principal component analysis (PCA) model was constructed and subsequently hierarchical cluster analysis (HCA) indicated the presence of two main groups, which were found to be independent of the populations to which the samples belong. Classes, based on the HCA class identifiers, were subsequently assigned to the samples, and an orthogonal projection to latent structures-discriminant analysis (OPLS-DA) model was then constructed, (R2 cum = 0.996 and Q2 cum = 0.967). The corresponding loadings plot allowed sericic acid, sericoside and resveratrol-3-rutinoside to be identified as biomarkers associated with the first group. Quantitative, rather than qualitative differences were responsible for the observed clustering pattern. Techniques that could be applied in quality control protocols for T. sericea root were investigated. High performance thin layer chromatography (HPTLC) analysis of the root extracts was optimised by testing different developing solvents and visualization reagents. The presence of the sericic acid (Rf = 0.80), sericoside (Rf = 0.49) and resveratrol-3-rutinoside (Rf = 0.36) were clearly visible on the plates. There were visible variations in the concentrations of resveratrol-3-rutinoside in representative samples from the 10 populations, corresponding to the UPLC results. The powdered samples were then analysed by mid-(MIR) infrared spectroscopy. Chemometric analysis of the data revealed no definitive clustering pattern. Partial least squares-discriminant analysis (PLS-DA) calibration models were established from the MIR spectral data combined with the accurate UPLC-values, for the prediction of the sericoside (R2Y = 0.848, Q2 = 0.757, RMSEP = 2.70 mg/g) and resveratrol-3-rutinoside (R2Y = 0.794, Q2 = 0.695, RMSEP = 4.37 mg/g) concentrations in powdered root samples. The antibacterial activities of the root extracts, column fractions and isolated compounds were determined using three Gram-positive and five Gram-negative bacteria, all selected due to their ability to cause intestinal and skin disorders. Extracts and fractions containing high concentrations of sericic acid exhibited the highest activities against Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 27858), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12223) and Shigella sonnei (ATCC 9292). The pure compound (sericic acid) was highly active against S. sonnei (MIC 0.078 μg/mL), a Gram- negative bacterium. There were no variations in the activity of the crude extracts against B. cereus and P. aeruginosa, while the MIC values obtained against S. typhi were variable and ranged from 0.25 to 1.0 mg/mL. Sericoside and resveratrol-3-rutinoside did not display any activity. The anti-oxidant activities were evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) and reducing power assays. The anti-oxidant assays revealed that resveratrol-3- rutinoside exhibited lower activity (DPPH = 186 μg/mL; RP = 184 μg/mL) compared to the crude extract (DPPH = 22.3 μg/mL; RP = 24.4 μg/mL) and ascorbic acid (DPPH = 11.3 μg/mL, RP = 145 μg/mL). Sericic acid and sericoside did not display any anti- oxidant activities. The variation in the anti-oxidant activities (4.58 to 26.0 μg/mL) of the samples from different populations was an indication of chemical variability. A toxicity study of the raw powdered plant material was conducted using vervet monkeys (Chlorocebus pygerythrus). Biochemical analysis (liver function tests, kidney function tests and hematology), physical and physiological examinations were conducted. The subjects were fed a normal diet supplemented with T. sericea root powder (2.14 g/kg per day) for 120 days, where after the diet was returned to normal (washout) for another 30 days. The treatment groups presented with elevated serum enzymes at Week 4, followed by the reduction of the elevated serum enzymes levels at Week 12. These results indicate short-term hepatotoxic effects, followed by hepatoprotective activity. Reduction of the serum glucose at Week 4 suggests hypoglycemic potential. However, elevated serum creatinine levels indicated possible nephrotoxicity. In conclusion, this study has indicated the variability in the chemical constituents of the roots of T. sericea, which affects the antibacterial and anti-oxidant activities. Sericic acid, resveratrol-3-rutinoside, and sericoside were, for the first time, identified as biomarkers that can be used for the quality control of raw root material to be used in herbal products. Sericic acid was also found to be the main antibacterial constituent of the roots. The hepatoprotective, nephrotoxic and hematotoxic effects observed in monkeys to which the root powder had been administered is cause for concern. / NRF