The antibacterial activity of extracts of nine plant species with good activity against Escherichia coli against five other bacteria and cytotoxicity of extracts.

BACKGROUND: The development of antibiotic resistant bacteria stems from a number of factors, including inappropriate use of antibiotics in human and animal health and their prolonged use as growth promoters at sub-clinical doses in poultry and livestock production. We were interested in investigating plants that could be useful in protecting humans or animals against diarrhoea. We decided to work on extracts of nine plant species with good activity against Escherichia coli based on earlier work in the Phytomedicine Programme. Leaves of nine medicinal plant species with high antibacterial activity against Escherichia coli were extracted with acetone and their minimal inhibitory concentration (MIC) values determined using a microplate serial dilution technique against Gram-positive (Staphylococcus aureus, Enterococcus faecalis and Bacillus cereus) and Gram-negative (Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa) bacteria. Bioautography was used to determine the number of bioactive compounds in each extract. In vitro safety of the extracts was determined using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay on Vero cells. RESULTS: The extracts were active against all the pathogens with average MICs ranging from 0.02 to 0.52 mg/ml. As expected E. coli was relatively sensitive, while E. faecalis and S. Typhimurium were more resistant to the extracts (average MICs of 0.28 mg/ml and 0.22 mg/ml respectively). Cremaspora triflora and Maesa lanceolata leaf extracts had higher activity than the other extracts against Gram-positive and Gram-negative pathogens with mean MICs of 0.07 mg/ml and 0.09 mg/ml respectively. Extracts of Maesa lanceolata and Hypericum roeperianum had the highest total antibacterial activity (TAA) at 1417 and 963 ml/g respectively. All extracts with the exception of that of Maesa lanceolata, Elaeodendron croceum and Calpurnia aurea had relatively low cytotoxicity with LC50 > 20 µg/ml. Cremaspora triflora had the best selectivity index (SI) against S. aureus and E. coli of 2.87 and 1.15 respectively. Hypericum roeperianum had a SI of 1.10 against B. cereus. Bioautography revealed 1-6 visible antimicrobial compounds that were generally non-polar. CONCLUSIONS: There was a weak positive, but statistically non-significant correlation between the potency of the extracts and their cytotoxicity (R = 0.45, ρ > 0.05). The activity of the extracts on the test bacteria was in some cases not correlated with cytotoxicity, as shown by selectivity indices >1. This means that cellular toxicity was probably not due to compounds with antibacterial activity. Some of the extracts had a good potential for therapeutic use against the bacterial pathogens or for application in treating diarhoea. It does not appear that activity against E. coli is a good predictor of activity against Gram-negative rather than Gram-positive bacteria. Further investigation is in progress on C. triflora and H. roeperianum, both of which had promising activities and potential safety based on cytotoxicity.

Acetone leaf extracts of some South African trees with high activity against Escherichia coli also have good antimycobacterial activity and selectivity index.

BACKGROUND: Tuberculosis is a world-wide problem affecting humans and animals. There is increasing development of resistance of the pathogens to current antimycobacterial agents. Many authors have investigated activities of extracts and isolated compounds from plants. The traditional uses of plants have frequently been the criterion to select plants investigated. In this contribution, we investigate whether plant extracts with very good activity against Escherichia coli may also be active against mycobacteria. METHODS: The antimycobacterial activity of eight South African tree leaf extracts with high activity against Escherichia coli were determined in vitro against Mycobacterium smegmatis, M. fortuitum and M. aurum, using a serial microdilution method. The cellular cytotoxicity was also determined by the MTT assay using Vero monkey kidney cells. The selectivity index was determined by dividing the cytotoxicity of extracts by MIC. RESULTS: The antimycobacterial activity of the extracts ranged from 0.02 to 2.5 mg/ml. Mycobacterium smegmatis was more sensitive to the extracts (Average MIC = 0.96 mg/ml) and Mycobacterium aurum was comparatively resistant (Average MIC = 2.04 mg/ml). The extracts of Cremaspora triflora had strong antimycobacterial activity with a MIC of 0.05 mg/ml that compared reasonably well with that of streptomycin (0.01 mg/ml) and rifampicin (0.03 mg/ml), p > 0.05. Cremaspora triflora had the best selectivity index of 2.87 against Mycobacterium fortuitum. CONCLUSION: The high activity of C. triflora extracts against the fast-growing mycobacteria and good cellular safety is promising. It may be interesting to investigate extracts against pathogenic M. tuberculosis, M. bovis and M. avium cultures and to isolate active antimycobacterial compounds.

Potency and selectivity indices of acetone leaf extracts of nine selected South African trees against six opportunistic Enterobacteriaceae isolates from commercial chicken eggs.

BACKGROUND: The rise in antimicrobial resistance in a plethora of nosocomial and opportunistic bacterial pathogens often isolated from commercial eggs, poses a serious public health concern. The existence of these contaminants may also serve as a drawback in the current efforts of improving the well-being of immunocompromised patients. The aim of this study was to determine the efficacy of plant extracts that had good activity on Escherichia coli in previous word on pathogens isolated from eggs for possible use in combating pathogens from eggs. METHODS: Acetone leaf extracts of nine trees with good activities against Escherichia coli were tested for their in vitro antibacterial activity against six opportunistic bacterial isolates from commercial eggs (Stenotrophomonas maltophilia, Klebsiella pneumoniae, Salmonella serotype Typhimurium, Proteus mirabilis, Enterobacter cloacae and Escherichia coli) using a serial microdilution method with tetrazolium violet as indicator of growth. Cytotoxicity was determined using a tetrazolium-based colorimetric assay against Vero kidney cells, and selectivity index calculated. RESULTS: The MIC values range of the different extracts against Stenotrophomonas maltophilia was 0.08-0.31 mg/ml, Klebsiella pneumonia 0.08-0.63 mg/ml, Salmonella ser. Typhimurium 0.08-0.63 mg/ml, Proteus mirabilis 0.02-1.25 mg/ml, Enterobacter cloacae 0.08-0.31 mg/ml and Escherichia coli 0.08-0.16 mg/ml respectively. Escherichia coli was the most sensitive while Proteus mirabilis was most resistant pathogen to the different test extracts, with mean MIC values of 0.08 mg/ml and 0.46 mg/ml respectively. Cremaspora triflora extracts had good activity against all the pathogenic egg isolates, with the exception of Proteus mirabilis. Maesa lanceolata and Elaeodendron croceum had the best total antibacterial activity (TAA), while generally the selectivity index of the extract was low (SI < 1). CONCLUSION: The exceptional activity of C. triflora extracts suggests that the plant has potential as a therapeutic agent against some members of the Enterobacteriaceae. Further pharmacological investigations may be interesting in the search for new antimicrobial leads.

The efficacy and safety of nine South African medicinal plants in controlling Bacillus anthracis Sterne vaccine strain.

BACKGROUND: Anthrax is a zoonotic disease caused by Bacillus anthracis, a Gram-positive spore-forming bacterium. The presence of the bacteria and the toxins in the blood of infected hosts trigger a cascade of pathological events leading to death. Nine medicinal plants with good activities against other bacteria were selected to determine their in vitro antibacterial activity against Bacillus anthracis Sterne strain. The cytotoxicity of the extracts on Vero kidney cells was also determined. RESULTS: The minimum inhibitory concentration (MIC) values of the extracts against Bacillus anthracis Sterne strain ranged from 0.02 to 0.31 mg/ml. Excellent MIC values were observed for the following plant species: Maesa lanceolata (0.02 mg/ml), Bolusanthus speciosus, Hypericum roeperianum, Morus mesozygia (0.04 mg/ml) and Pittosporum viridiflorum (0.08 mg/ml). The total antibacterial activity of the extracts ranged from 92 to 5562 ml/g. Total activity presents the volume to which the extract from 1 g of plant material can be diluted and still inhibit microbial growth. Maesa lanceolata and Hypericum roeperianum had the highest total activity with values of 5562 and 2999 ml/g respectively. The extracts of Calpurnia aurea had the lowest total activity (92 ml/g). The cytotoxicity determined on Vero cells indicated that most of the extracts were relatively non-toxic compared to doxorubicin (LC50 8.3 ± 1.76 µg/ml), except for the extracts of Maesa lanceolata, Elaeodendron croceum and Calpurnia aurea with LC50 values at 2.38 ± 0.25, 5.20 ± 0.24 and 13 ± 2.26 µg/ml respectively. The selectivity index (SI) ranged from 0.02 to 1.66. Hypericum roeperianum had the best selectivity index, (SI = 1.66) and Elaeodendron croceum had lowest value (SI = 0.02). CONCLUSIONS: The crude acetone extracts of the selected plant species had promising antibacterial activity against Bacillus anthracis. Maesa lanceolata extracts could be useful as a disinfectant and Hypericum roeperianum could be useful to protect animals based on its high total activity and selectivity index. Further investigation of these plant extracts may lead to the development of new therapeutic agents to protect humans or animals against anthrax.

The anti-arthritic, anti-inflammatory, antioxidant activity and relationships with total phenolics and total flavonoids of nine South African plants used traditionally to treat arthritis.

BACKGROUND: Oxidative stress predisposes the human and animal body to diseases like cancer, diabetes, arthritis, rheumatoid arthritis, atherosclerosis and chronic inflammatory disorders. Hence, this study seeks to determine the antioxidant, anti-inflammatory and anti-arthritic activities of acetone leaf extracts of nine South African medicinal plants that have been used traditionally to treat arthritis and inflammation. METHODS: The anti-inflammatory activity of the extracts was determined by investigating inhibition of nitric oxide production in lipopolysaccharide activated RAW 264.7 macrophages as well as 15-lipoxygenase enzyme inhibition. An anti-protein denaturation assay was used to determine the anti-arthritic properties of the extracts. The antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) radical scavenging assays and ferric reducing antioxidant power (FRAP). The total phenolic and total flavonoid concentration of extracts were determined by using standard methods. RESULTS: All extracts inhibited nitric oxide production in a dose-dependent manner in the LPS-stimulated RAW 264.7 macrophages. Extracts of Maesa lanceolata and Heteromorpha arborescens inhibited NO production by 99.16 % and 89.48 % at a concentration of 30 µg/ml respectively. Elaeodendron croceum and Calpurnia aurea extracts had strong activity against 15-lipoxygenase activity with IC50 values of 26.23 and 34.70 µg/ml respectively. Morus mesozygia and Heteromorpha arborescens extracts had good in vitro anti-arthritic activity with IC50 values of 11.89 and 53.78 µg/ml, the positive control diclofenac sodium had IC50 value of 32.37 µg/ml. The free radical scavenging activity of the extracts in DPPH assays ranged between 7.72 and 154.77 µg/ml. Trolox equivalent antioxidant capacity (TEAC) and FRAP values ranged from 0.06 to 1.32 and 0.06 to 0.99 respectively. CONCLUSIONS: Results from this study support the traditional use of the selected medicinal plants in the management of arthritis and other inflammatory conditions. The free radical scavenging capacity of the extracts may be related to an immune boosting potential.

Predicting the influence of multiple components on microbial inhibition using a logistic response model--a novel approach.

BACKGROUND: There are several synergistic methods available. However, there is a vast discrepancy in the interpretation of the synergistic results. Also, these synergistic methods do not assess the influence the tested components (drugs, plant and natural extracts), have upon one another, when more than two components are combined. METHODS: A modified checkerboard method was used to evaluate the synergistic potential of Heteropyxis natalensis, Melaleuca alternifolia, Mentha piperita and the green tea extract known as TEAVIGO™. The synergistic combination was tested against the oral pathogens, Streptococcus mutans, Prevotella intermedia and Candida albicans. Inhibition data obtained from the checkerboard method, in the form of binary code, was used to compute a logistic response model with statistically significant results (p < 0.05). This information was used to construct a novel predictive inhibition model. RESULTS: Based on the predictive inhibition model for each microorganism, the oral pathogens tested were successfully inhibited (at 100% probability) with their respective synergistic combinations. The predictive inhibition model also provided information on the influence that different components have upon one another, and on the overall probability of inhibition. CONCLUSIONS: Using the logistic response model negates the need to 'calculate' synergism as the results are statistically significant. In successfully determining the influence multiple components have upon one another and their effect on microbial inhibition, a novel predictive model was established. This ability to screen multiple components may have far reaching effects in ethnopharmacology, agriculture and pharmaceuticals.

Biological Activities of Heteropyxis natalensis Against Micro-Organisms Involved in Oral Infections.

The use of complementary and alternative medicine from plants in South Africa, as in the rest of the world, continues to increase. Heteropyxis natalensis, known as the Lavender tree, is indigenous to South Africa and is traditionally used for oral care. The ethanolic extract, of the leaves and twigs, of H. natalensis was investigated for antimicrobial activity against selected oral microorganisms. Actinomyces israelii was found to be the most sensitive oral microorganism to the extract, with a minimum inhibitory concentration (MIC) of 0.88 mg/ml and an MIC of 2.6 mg/ml against Streptococcus mutans. Five known compounds were identified from the ethanolic extract of H. natalensis. The compounds were identified as aurentiacin A (1), cardamomin (2), 5-hydroxy-7-methoxy-6-methylflavanone (3), quercetin (4) and 3,5,7-trihydroxyflavan (5). The MICs of the compounds 1 and 4 were found to be 0.06 mg/ml and 1 mg/ml, respectively, against A. israelii. The cytotoxicity, acute and sub-acute toxicity in pre-clinical studies were also determined for H. natalensis. The extract showed moderate cytotoxicity (35.56 ± 0.16 µg/ml) on human monocyte cells. The acute and sub-acute toxicity analysis of H. natalensis indicated the NOEL (no-observed-effect level) at 200 mg/kg. Interleukin-8 (IL-8) is a chemokine that stimulates the recruitment of leukocytes. A significant reduction of IL-8 production by macrophage cells was observed when exposed to the extract of H. natalensis. It is possible that H. natalensis can prevent excessive tissue damage in periodontal diseases through its reduction of inflammation. Enzymatic bioanalysis of lactic and acetic acid production from Streptococcus mutans and Lactobacillus paracasei was done. A reduction in the acid production from each bacterium was observed on exposure to the extract of H. natalensis. Consequently, this increased the pH, which could possibly reduce the demineralization of enamel which may help prevent the formation of dental caries. In addition the extract may be considered for preventing periodontal diseases.

The use of Lespedeza cuneata for natural control of gastrointestinal nematodes in Merino sheep.

Lespedeza cuneata (poorman's lucerne; sericea lespedeza), a tannin-rich perennial legume, was offered as hay to dry Merino ewes in a confined feeding experiment to evaluate the effect on the level of gastrointestinal parasite infection in sheep. Medicago sativa (a low tannin containing perennial legume) was used as the control treatment. Parameters faecal egg count (FEC), FAMACHA© scores and rectal temperatures were used. FECs were substantially lower (p = 0.05) in the Lespedeza group after 35 days, together with a trend of higher rectal temperatures, compared with the Medicago group. Although non-significant (p > 0.05), the higher rectal temperatures suggested a lower level of anaemia in the sheep on the Lespedeza ration and, therefore, a lower parasite-worm burden. However, FAMACHA