A.R. Ndhlala

Natural Antioxidants: Fascinating or Mythical Biomolecules?

Research on the use, properties, characteristics and sources of antioxidants especially phenolic compounds, flavonoids, vitamins, synthetic chemicals and some micronutrients began in the late 18th century. Since then antioxidant research has received considerable attention and over a hundred thousand papers have been published on the subject. This has led to a rampant use of antioxidants in order to try to obtain and preserve optimal health. A number of nutraceuticals and food supplements are frequently fortified with synthetic or natural antioxidants. However, some research outcomes have led to the belief that antioxidants exist as mythical biomolecules. This review provides a critical evaluation of some common in vitro antioxidant capacity methods, and a discussion on the role and controversies surrounding non-enzymatic biomolecules, in particular phenolic compounds and non-phenolic compounds, in oxidative processes in an attempt of stemming the tidal wave that is threatening to swamp the concept of natural antioxidants.

Anti-inflammatory, anticholinesterase, antioxidant and phytochemical properties of medicinal plants used for pain-related ailments in South Africa.

ETHNOPHARMACOLOGICAL RELEVANCE Extracts of seven South African medicinal plants used traditionally for the treatment of pain-related ailments were evaluated. AIMS OF THE STUDY The study was aimed at evaluating medicinal and therapeutic potentials of the investigated traditional medicinal plants. Plant extracts were evaluated for anti-inflammatory activity and other pharmacological properties such as anticholinesterase and antioxidant activities. Phytochemical analysis of total phenolic contents, condensed tannins, gallotannins and flavonoids in the aqueous methanol extracts of the medicinal plants were also carried out. MATERIALS AND METHODS The evaluation of anti-inflammatory activity of 50% methanol (50% MeOH), petroleum ether (PE), dichloromethane (DCM) and ethanol (EtOH) plant extracts was done against cyclooxygenase-1 and -2 (COX-1 and COX-2) enzymes. 50% MeOH, PE, DCM and EtOH extracts were tested for acetylcholinesterase (AChE) inhibition, while 50% MeOH extracts were tested for 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging activity and ferric-reducing power in the antioxidant assays. Total phenolic compounds, condensed tannins, gallotannins and flavonoids were quantitatively determined using spectrophotometric methods. RESULTS At the screening assay concentration (0.25 microg/microl), 13 extracts showed good COX-1 inhibitory activity (>50%), while good activity was observed in 15 extracts against COX-2 enzyme. All the extracts of Crinum moorei (bulbs) showed good inhibition against both COX-1 and COX-2 enzymes. Though not significantly different (P=0.05), the highest COX-1 percentage inhibition (100%) was shown by Aloe ferox leaf PE and Colocasia antiquorum tuber DCM extracts, while Colocasia antiquorum tuber PE extract exhibited the highest (92.7%) percentage inhibition against COX-2. Crinum moorei bulb DCM extract showed the lowest EC(50) value (2.9 microg/ml) in the AChE assay. In addition, good to moderate bioactivities were observed in some extracts of Aloe ferox (leaves), Crinum moorei (bulbs) and Pycnostachys reticulata (leaves) in all the assays. The presence and/or amounts of phenolic compounds varied with plant species. CONCLUSION The results obtained in this study validate the use of the investigated medicinal plants in South African traditional medicine for pain-related ailments.

Antimicrobial properties and phenolic contents of medicinal plants used by the Venda people for conditions related to venereal diseases.

ETHNOPHARMACOLOGY RELEVANCE Many people are infected by venereal diseases and the human immunodeficiency virus (HIV) in rural areas. Sexual transmitted diseases are considered a disgrace in the community because of the stigmas attached to them. Indigenous people tend to use several medicinal plants to treat these infectious diseases rather than western medicines. AIM OF THE STUDY This study was aimed to evaluate the antibacterial, antifungal, antigonococcal, HIV-type 1 reverse transcriptase (RT) and to determine phenolic content of twelve medicinal plants used by the Venda people to treat venereal diseases. MATERIALS AND METHODS The dried plant materials of twelve medicinal plants were extracted with petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water. The extracts were evaluated for their antimicrobial properties against two Gram-positive (Bacillus subtilis and Staphylococcus aureus), three Gram-negative (Neisseria gonorrhoeae, Escherichia coli and Klebsiella pneumoniae) bacteria and a fungus Candida albicans. The phenolic contents including total phenolics, flavonoids, gallotannins and condensed tannins of the methanolic extracts of the same plants were also determined. RESULTS DCM and EtOH extracts of Bolusanthus speciosus bark and stems exhibited good activity (<0.1mg/ml) against Escherichia coli and Staphylococcus aureus with MIC values ranging from 0.098 to 0.012 mg/ml. The DCM extracts of Bolusanthus speciosus bark showed the best activity against Candida albicans with MIC and MFC values of 0.012 mg/ml. Water and 50% methanol extracts of Acacia karroo bark, Bolusanthus speciosus stems and Ximenia caffra roots and leaves showed good RT inhibition percentages (>70%) at 1mg/ml. All tested extracts exhibited dose dependent IC(50) values ranging from (0.1 to 0.6 mg/ml). Almost all plant species investigated contained phenolic compounds, which were greater than 5mg/g with the exception of Adansonia digitata bark and Aloe chabaudii roots. The highest level of flavonoids (11.9 μg/g) were detected in Ximenia caffra leaves. Whereas the highest amount of gallotannins were detected in Ekebergia capensis bark (69 μg/g). Condensed tannins were higher in Ekebergia capensis bark and Ximenia caffra roots (0.47 and 0.48% respectively). Adansonia digitata bark and Aloe chabaudii roots exhibited low levels of phenolic compounds as well as antimicrobial activities. CONCLUSION The presented results offer supporting evidence for effective use of selected plant extracts. More in vitro confirmatory tests using other assays and/or in vivo tests are required. Toxicity studies are urgently needed to support the safe usage of these plants.

Anti-inflammatory and phytochemical properties of twelve medicinal plants used for treating gastro-intestinal ailments in South Africa

ETHNOPHARMACOLOGICAL RELEVANCE The investigated medicinal plants are commonly used for the treatment of pains and cramps related to gastro-intestinal tract infections in South African traditional medicine. AIMS OF THE STUDY This study aimed to evaluate the ability of the plant extracts to inhibit cyclooxygenase enzymes. Phytochemical analysis was also carried out in the quest to determine some plant metabolites that may be responsible for the observed anti-inflammatory activity. MATERIALS AND METHODS The cyclooxygenase assay was used to test for the anti-inflammatory activity of the plant extracts using cyclooxygenase-1 and -2 (COX-1 and COX-2) enzymes. Total phenolic compounds including condensed tannins, gallotannins and flavonoids were quantitatively determined using spectrophotometric methods. Qualitative tests for alkaloids and saponins were also carried out. RESULTS Most of the plant extracts evaluated showed dose dependent activity against COX-1 and/or COX-2 enzymes. Agapanthus campanulatus root dichloromethane extract showed the highest COX-2 inhibitory activity (83.7%) at 62.5 microg/ml. The presence and/or amounts of phenolics, condensed tannins, gallotannins, flavonoids, alkaloids and saponins varied with plant parts and species. CONCLUSION The results support the use of the investigated plant in treating pain and cramp related to gastro-intestinal tract infections. To some extent, the observed anti-inflammatory activity could be attributed to the various plant secondary metabolites detected in the plant materials.

In vitro pharmacological effects of manufactured herbal concoctions used in KwaZulu-Natal South Africa

ETHNOPHARMACOLOGY RELEVANCE Manufactured and packaged herbal mixtures, known locally as muthi concoctions, have become a new feature of traditional medicine in South Africa. These herbal concoctions are often claimed to cure several diseases ranging from minor illness to life threatening conditions. AIM OF THE STUDY To document the prevalence, variety and evaluate the efficacy of selected herbal concoctions sold in KwaZulu-Natal. MATERIALS AND METHODS Nine herbal concoctions were evaluated for their effects as antibacterial and antifungal agents as well as their ability to inhibit the cyclooxygenase (COX-1 and COX-2) enzymes using standard in vitro methods. RESULTS Imbiza ephuzwato and Ibhubezi, both multipurpose mixtures showed high abilities to inhibit bacterial and fungal growth as well as inhibit the COX-1 and COX-2 enzymes. Umzimba omubi and Umuthi wekukhwehlela ne zilonda, both remedies for wounds, boils and chest infections were not active against the two Gram-positive and two Gram-negative bacterial strains tested. Vusa umzimba and Supreme one hundred were active against Candida albicans. CONCLUSIONS Four of the herbal concoctions showed no activity in all of the assays tested while only two showed good activity. Activity cannot be ruled out as there are many mechanisms upon which natural products can act.

Plant composition, pharmacological properties and mutagenic evaluation of a commercial Zulu herbal mixture: Imbiza ephuzwato

ETHNOPHARMACOLOGY RELEVANCE Imbiza ephuzwato is a traditional herbal tonic made from a mixture of extracts of roots, bulbs, rhizomes and leaves of 21 medicinal plants and is used in traditional medicine as a multipurpose remedy. AIM OF THE STUDY To compile and investigate the bioactivity and mutagenic effects of extracts of the 21 plant species used in the preparation of Imbiza ephuzwato herbal tonic. MATERIALS AND METHODS The 21 plant species used to make Imbiza ephuzwato herbal mixture were each investigated for their pharmacological properties. Petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water extracts of the 21 plants were evaluated against two gram-positive, two gram-negative bacteria and a fungus Candida albicans. The extracts were also evaluated for their inhibitory effects against cyclooxygenase (COX-1 and -2) and acetylcholinesterase AChE enzymes. Mutagenic effects of the water extracts were evaluated using the Ames test. RESULTS Gunnera perpensa and Rubia cordifolia were the only plant species used to manufacture Imbiza ephuzwato that had water extracts which showed good antibacterial activity. The extracts of G. perpensa (EtOH), Hypericum aethiopicum (DCM) and Urginea physodes (EtOH) showed the best antifungal activity. The water extracts of H. aethiopicum, G. perpensa, Drimia robusta, Vitellariopsis marginata, Scadoxus puniceus and Momordica balsamina showed percentage inhibition of COX-1 that was over 70%. For COX-2 enzyme, the water extracts of G. perpensa, Cyrtanthus obliquus, M. balsamina and Tetradenia riparia exhibited inhibitory activity above 70%. Water extracts of G. perpensa, C. obliquus, V. marginata, Asclepias fruticosa and Watsonia densiflora showed good AChE inhibitory activity (>80%). The Ames test results revealed that all the water extracts of the 21 plant species used to make Imbiza ephuzwato were non-mutagenic towards the Salmonella typhimurium TA98 strain for the assay with and without S9 metabolic activation. In contrast, Imbiza ephuzwato showed mutagenic effects after exposure to S9 enzyme mixture. CONCLUSION The observed activities of some plant extracts, if supported by other confirmatory tests, may justify their inclusion in the makeup of Imbiza ephuzwato herbal mixture as well as their use in traditional medicine. Further studies aimed at investigating possible synergistic effects as a result of mixing plant extracts are necessary. The reported mutagenicity in Imbiza ephuzwato could be as a result of interaction of biomolecules in the heterogeneous mixture, yielding compounds that are converted to mutagenic agents by xenobiotic metabolizing enzymes. It is therefore important to carry out further studies aimed at identifying and eliminating the sources of the mutagenic compounds in the heterogeneous mixture.

Antimicrobial, anti-inflammatory and mutagenic investigation of the South African tree aloe (Aloe barberae)

ETHNOPHARMACOLOGY RELEVANCE In recent times, many products ranging from aloe drinks to aloe gels, powders, capsules, and creams have appeared on the commercial market prepared from different aloe species including Aloe barberae. These products are used in ethnomedicine to treat various conditions including gastrointestinal disorders, insect bites, skin burns and other skin injuries by traditional communities. AIM OF THE STUDY This study was aimed at evaluating the antibacterial, antifungal and anti-inflammatory activities as well as genotoxic effects of different extracts of Aloe barberae. MATERIALS AND METHODS Organic and water extracts of the upper stem, young bark, mature bark, leaves and roots of the South African tree aloe (Aloe barberae) were evaluated for their antimicrobial [gram-positive (Bacillus subtilis and Staphylococcus aureus), gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria as well as the fungus Candida albicans], anti-inflammatory (COX-1 and COX-2) and mutagenic properties (Ames test). Thin layer chromatography (TLC) was used to compare the phytochemical profiles of different extracts of Aloe barberae. RESULTS The petroleum ether (PE) and dichloromethane (DCM) extracts of the mature bark, leaves and roots exhibited good activity against all the bacteria and fungus Candida albicans with minimum inhibitory concentrations (MIC) ranging from 0.195 to 1.56 mg/ml. All the PE extracts evaluated showed a high activity (>70%) in both COX-1 and COX-2 assays. Apart from the organic extracts of the root with consistently good activity (>70%), all the remaining extracts showed moderate activity (40-69%) in COX-1 assay. The PE extracts also showed a dose dependent increase in activity. Ultraviolet (UV) spectrum of the leaves and root EtOH extracts indicated the presence of compounds that could absorb UV light (wavelength: 190-820 nm). None of the extracts had a mutagenic effect in the Salmonella/microsome assay against a tester strain, TA98. CONCLUSION Activity observed in the bark, leaves and roots of Aloe barberae validates its use in commercial herbal products, ethnobotany and ethnoveterinary medicine by South African communities and small scale farmers to treat various conditions.

Pharmacological properties and protein binding capacity of phenolic extracts of some Venda medicinal plants used against cough and fever

ETHNOPHARMACOLOGY RELEVANCE Several ailments are caused by infectious bacteria and in other diseases; they act as co-infection which complicate human life by causing health hazards. In Venda (South Africa), many plants are used in traditional medicine to treat cough and fever. AIM OF THE STUDY This study was aimed at evaluating the antibacterial and antifungal properties, cyclooxygenases (COX), acetylcholinesterase (AChE) enzyme inhibitory effects and the phenolic composition as well as mutagenic properties of six medicinal plants used by the Venda people of Limpopo Province of South Africa against cough and fever. MATERIALS AND METHODS The petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water extracts of six plants were tested against four infectious bacteria (Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus) and a fungus Candida albicans. The same extracts were evaluated for their ability to inhibit COX-1 and -2 enzymes. Methanolic and water extracts of the same plant were tested for acetylcholinesterase inhibitory effects. Total phenolics, flavonoids, gallotannins and condensed tannins were determined. The ability of the extracts to bind and precipitate proteins was also investigated. The extracts were investigated for genotoxicity with and without S9 (metabolic activation) against three Salmonella typhimurium tester strains TA98, TA100 and TA102. RESULTS The organic extracts of Rhus lancea leaves exhibited the best antibacterial activity with minimum inhibitory concentration (MIC) values ranging from 0.0061 to 0.049mg/ml. The best antifungal activity was observed from a DCM extract of Syzygium cordatum leaves with a MIC value of 0.195mg/ml. The methanolic and water extracts of the same plant exhibited high inhibitory effects towards AChE with IC(50) values of 0.22 and 0.26mg/ml, respectively. The highest levels of flavonoids and gallotannins were detected in Spirostachys africana bark; 11.57 and 48.88μg/g, respectively. The highest percentages (1.2%) of condensed tannins were detected in Uvaria caffra leaves. The high levels of phenolic compounds may have been responsible for high antimicrobial activities for extracts of S. africana bark and U. caffra leaves. S. cordatum leaves represented the highest affinity for protein binding with 93%. All the extracts were non-mutagenic towards the three tested strains with and without S9 metabolic activation. CONCLUSION The result obtained in this study goes a long way in validating the ethnobotanical usage of these medicinal plants in the treatment of cough and fever by the Venda people. However, more evidence obtainable from other assays not performed here are urgently required to confirm these results.

Antimicrobial and Selected In Vitro Enzyme Inhibitory Effects of Leaf Extracts, Flavonols and Indole Alkaloids Isolated from Croton menyharthii

Croton species are used in folk medicine in the management of infections, inflammation and oxidative stress-related diseases. In order to isolate, characterize and evaluate the bioactive constituents of Croton menyharthii Pax leaf extracts, repeated column fractionation of the ethyl acetate fraction from a 20% aqueous methanol crude extract afforded three flavonols identified by NMR (1D and 2D) spectroscopic methods as myricetrin-3-O-rhamnoside (myricetrin, 1), quercetin-3-O-rhamnoside (2) and quercetin (3) along with an indole alkaloid, (E)-N-(4-hydroxycinnamoyl)-5-hydroxytryptamine, [trans-N-(p-coumaroyl) serotonin, 4]. All the compounds are reported from the leaf extract of this plant for the first time. The crude extracts, four solvent fractions (hexane, DCM, ethyl acetate and butanol) and isolated compounds obtained from the leaves were evaluated for their inhibitory effects on selected bacteria, a fungus (Candida albicans), cyclooxygenase (COX-2), α-glucosidase and acetylcholinesterase (AChE). Amongst the compounds, quercetin (3) was the most active against Bacillus subtilis and Candida albicans while myricetrin-3-O-rhamnoside (1) and trans-N-(p-coumaroyl) serotonin (4) were the most active compounds against Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus. The inhibitory activity of myricetrin-3-O-rhamnoside (1) against COX-2 was insignificant while that of the other three compounds 2–4 was low. The AChE inhibitory activity of the alkaloid, trans-N-(p-coumaroyl) serotonin was high, with a percentage inhibitory activity of 72.6% and an IC50 value of 15.0 µg/mL. The rest of the compounds only had moderate activity. Croton menyharthii leaf extracts and isolated compounds inhibit α-glucosidase at very low IC50 values compared to the synthetic drug acarbose. Structure activity relationship of the isolated flavonols 1–3 is briefly outlined. Compounds 1–4 and the leaf extracts exhibited a broad spectrum of activities. This validates the ethnomedicinal use of the plant in folk medicine.

Ethnopharmacological study of plants from Pondoland used against diarrhoea.

ETHNOPHARMACOLOGICAL RELEVANCE Waterborne diseases such as diarrhoea are common world wide, including in Bizana, South Africa where the majority of rural dwellers depend largely on water from unprotected sources. The people from Bizana use medicinal plants as their first line of health care to cure and prevent diarrhoea. AIM OF THE STUDY To record and document plants used for the treatment of diarrhoea in Bizana, to evaluate antibacterial and anti-inflammatory activities of selected plant extracts as well as to perform genotoxicity testing of evaluated plants. MATERIALS AND METHODS An ethnobotanical approach was used to select plants used for treating diarrhoea in Bizana for pharmacological assays using questionnaires. Nine plants were selected for bioassays based on their frequency index and the fact that they have never been evaluated against diarrhoea causing-microorganisms. The petroleum ether (PE), dichloromethane (DCM), 70% ethanol (EtOH), and water extracts were evaluated for antibacterial (Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and Shigella flexneri) activity using the microdilution technique, their ability to inhibit COX-1 and COX-2 enzymes. Genotoxicity was evaluated using the Salmonella microsome assay. RESULTS This study revealed that 34 plant species belonging to 27 families are used for the treatment of diarrhoea in Bizana. The extracts showed good inhibitory activity with MIC values ranging from 0.39 to 12.5mg/ml. The best activity was exhibited by DCM extracts of Rapanea melanophloeos, and EtOH extracts of Ficus craterostoma and Maesa lanceolata with MIC values of 0.098mg/ml. The inhibitory activity against COX-1 enzyme was higher than COX-2, with 19 plant extracts for the former and 7 for the latter. All the tested plant extracts were not mutagenic at all concentrations tested against all tester strains of bacteria. CONCLUSION In view of the fact that the plants were selected based on their ethnobotanical usage for treating diarrhoea, the activities reported here goes a long way in validating the plants for traditional use.