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

Abstract

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.

Keywords: Antibacterial activity; Cellular safety; Correlation; Efficacy; Nosocomial bacteria; Potency.

Fig. 1

a Chromatogram developed in Benzene: Ethanol: Ammonia (BEA) solvent system of the different plant leaf acetone extracts sprayed with vanillin. b Bioautography of Staphylococcus aureus developed with BEA; white bands indicate compounds that inhibit the growth of the bacteria. HR = Hypericum roeperianum, CT = Cremaspora triflora, HA = Heteromorpha arborescens, PV = Pittosporum viridiflorum, BS = Bolusanthus speciosus, CA = Calpurnia aurea, ML = Maesa lanceolata, EC = Elaeodendron croceum, MM = Morus mesozygia

Fig. 2

Average MIC values of the nine-acetone leaf extracts against all the test bacteria; the lower the MIC values the most potent the extract. There is a significant difference between the MIC values of the different crude extracts against the test bacteria (ρ < 0.05). HR = Hypericum roeperianum, CT = Cremaspora triflora, HA = Heteromorpha arborescens, PV = Pittosporum viridiflorum, BS = Bolusanthus speciosus, CA = Calpurnia aurea, ML = Maesa lanceolata, EC = Elaeodendron croceum, MM = Morus mesozygia

Fig. 3

The mean MIC in mg/ml of the acetone leaf extracts of the nine plants against five different bacteria. SA = Staphylococcus aureus, EF = Enterococcus faecalis, BC = Bacillus cereus, EC = Escherichia coli, ST = Salmonella Typhimurium, PA = Pseudomonas aeruginosa, ^a = Enterococcus faecalis and Escherichia coli (ρ < 0.01), ^b = Salmonella Typhimurium and Escherichia coli (ρ < 0.01)

Fig. 4

The mean MIC in mg/mlof the acetone leaf extracts of nine plants against all the test bacteria (ρ < 0.05). HR = Hypericum roeperianum, CT = Cremaspora triflora, HA = Heteromorpha arborescens, PV = Pittosporum viridiflorum, BS = Bolusanthus speciosus, CA = Calpurnia aurea, ML = Maesa lanceolata, EC = Elaeodendron croceum, MM = Morus mesozygia, ^* = mean MIC value of H. arborescens is significantly higher than MIC of M. lanceolata (ρ < 0.01)

Fig. 5

Efficacy (mean TAA values, ml/g) of the different acetone extracts against all the test bacteria. The higher the TAA value, the more efficacious the plant. The quantity extracted from 1 g of CL can be diluted to 1417 ml and will still inhibit on average the different bacteria. HR = Hypericum roeperianum, CT = Cremaspora triflora, HA = Heteromorpha arborescens, PV = Pittosporum viridiflorum, BS = Bolusanthus speciosus, CA = Calpurnia aurea, ML = Maesa lanceolata, EC = Elaeodendron croceum, MM = Morus mesozygia