Avoiding pitfalls in determining antimicrobial activity of plant extracts and publishing the results

Abstract Background There is an urgent need to discover new antimicrobial compounds or extracts to address the crucial problem of increasing microbial resistance against current antibiotics. Plant chemical biodiversity is a valuable potential resource. Although compounds from plants are used as basis for several human drugs, no commercially successful antibiotic has yet been discovered from plants, despite more than a thousand publications in this field per year. This may be due to wrong methods that have been used or wrong plants that were investigated. A lot of energy is wasted by using techniques such as agar diffusion that do not work well with plant extracts. Many manuscripts are rejected before sending to reviewers because wrong methods are used. Antimicrobial activity of plant extracts based on agar diffusion studies have limited value. Methods Results obtained from several hundred of our publications in this area as researcher and experience as editor was used to identify difficulties in generating reproducible data. Other publications were also consulted and procedures used were evaluated. Results Because many of the antimicrobial compounds in plant extracts are relatively non-polar, these compounds do not diffuse well in the aqueous agar matrix used in agar diffusion studies. So many other factors also influence the zone of inhibition, that results between different laboratories are not comparable. The different methods used to determine the minimal inhibitory concentration (MIC) in serial dilution studies have been discussed. Using p-iodonitrotetrazolium violet to indicate growth provided the best results. Factors such as inoculum size, solvent, selection of positive controls and selection of plants to investigate also play a role. A method developed to determine antibacterial and antifungal activity of plant extracts work very well and is widely used based on > 1830 citations. Conclusions By using proposed methods manuscripts will provide reproducible information that may be published in good journals. The publications could contribute to a rational basis for finding compounds or extracts from plants that may address the problem of antimicrobial resistance. Random screening of a large number of plant species using this technique have already led to some commercial applications and identification of a potentially new antifungal framework compound

Repellent properties of Rotheca glabrum plant extracts against adults of Rhipicephalus appendiculatus

Abstract Background Rotheca glabrum (formerly known as Clerodendrum glabrum [Verbenaceae]) is used by local communities in the Limpopo Province of South Africa to control ticks on livestock and was selected from the database of the ARC-Onderstepoort Veterinary Institute. Its leaves were extracted using organic solvents ranging from polar to non-polar solvents (methanol, acetone and dichloromethane (DCM)). In addition, the traditional soap-water (infusion) and water-based (decoction) methods were used. The tick repelling activity was determined against the adult stage of the livestock tick Rhipicephalus appendiculatus. Results In the tick-climbing repellency bioassay a 30% acetone extract had a significant (p ≤ 0.05) repellent effect against adults of R. appendiculatus. The extract was still active at a lower concentration of 10%. The hexane fraction from the R. glabrum acetone extract had a higher tick repellency activity than the positive controls Amitix and Bayticol at the same concentrations. Unfortunately, the activity decreased after 2.5 h, probably due to volatility of the biologically active compound(s) within the extract. Conclusion Attempts were made to isolate the repellent compound from the acetone extract of R. glabrum. The process produced very good results up to a late stage in the bioassay-guided fractionation process. At that point, the repellent activity was lost. When two fractions were combined, the repellent activity was regained. These results provide strong evidence for the existence of a synergisticactivity of different compounds. It may be better to concentrate on extracts that would kill ticks rather than on extracts that would repel ticks

The antibacterial activity, antioxidant activity and selectivity index of leaf extracts of thirteen South African tree species used in ethnoveterinary medicine to treat helminth infections

BACKGROUND: Diseases caused by bacteria remain a major challenge globally and particularly in sub-Saharan Africa. The plants used in this study have been used in South Africa to treat helminth infections in livestock and humans. In a previous study we found a correlation between antifungal and anthelmintic activity in some cases. In this study we examined other potential uses of these thirteen plant species by determining the antibacterial and antioxidant activity of the leaf acetone extracts. The antibacterial activity was determined by using a serial microdilution method against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis. Bioautography was used to determine the number of antibacterial compounds. The antioxidant activity was determined using the ABTS and DPPH methods. RESULTS: Maesa lanceolata and Leucosidea sericea with an MIC of 0.02 mg/ml had excellent antibacterial activity against Enterococcus faecalis and Pseudomonas aeruginosa. There was a poor correlation between antioxidant activity and antibacterial activity with R(2) = 0.143. This is because antibacterial activity is mainly related to non-polar compounds and antioxidant activity to polar compounds. Maesa lanceolata extracts had a low cytotoxicity with a selectivity index of 5.2, 2.6, 2.6 and 1.3 for P. aeruginosa, E. faecalis, E. coli and S. aureus respectively. Strychnos mitis extracts had a therapeutic index of 1.1 for E. coli. CONCLUSIONS: This study shows that plant extracts of some species used in ethnoveterinary medicine as anthelmintic may also have excellent antibacterial activity

Efficacy and toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary medicine in South Africa on egg hatching and larval development of Haemonchus contortus

BACKGROUND: Helminthiasis is a major limitation to the livestock industry in Africa. Haemonchus contortus is the singular most important helminth responsible for major economic losses in small ruminants. The high cost of anthelmintics to small farmers, resistance to available anthelmintics and residue problems in meat and milk consumed by humans further complicates matters. The use of plants and plant extracts as a possible source of new anthelmintics has received more interest in the last decade. Our aim was not to confirm the traditional use, but rather to determine activity of extracts. Based on our past experience acetone was used as extractant. Because it is cheaper and more reproducible to evaluate the activity of plant extracts, than doing animal studies, the activity of acetone leaf extracts of thirteen plant species used traditionally in ethnoveterinary medicine in South Africa were determined using the egg hatch assay and the larval development test. Cytotoxicity of these extracts was also evaluated using the MTT cellular assay. RESULTS: Extracts of three plant species i.e. Heteromorpha trifoliata, Maesa lanceolata and Leucosidea sericea had EC(50) values of 0.62 mg/ml, 0.72 mg/ml and 1.08 mg/ml respectively for the egg hatch assay. Clausena anisata; (1.08 mg/ml) and Clerodendrum glabrum; (1.48 mg/ml) extracts were also active. In the larval development assay the H. trifoliata extract was the most effective with an EC(50) of 0.64 mg/ml followed by L. sericea (1.27 mg/ml). The activities in the larval development test were generally lower in most plant species than the egg hatch assay. Based on the cytotoxicity results C. anisata was the least toxic with an LC(50) of 0.17 mg/ml, while Cyathea dregei was the most toxic plant with an LC(50) of 0.003 mg/ml. The C. anisata extract had the best selectivity index with a value of 0.10 and 0.08 for the two assays, followed by H. trifoliata and L. sericea with values of 0.07, 0.07 and 0.05, 0.04. The C. dregei extract had the worst selectivity index with a value of 0.00019 for both assays. CONCLUSION: The result of this study indicates which species should be further investigated in depth for isolation of compounds

Biological activity of folkloric plants used in the treatment of ‘u wela’ against pathogens

Background: ‘U wela’ also known as ‘Divhu’ in ‘Tshivenda’ is a sexually transmitted infection caused by a combination of fungal and bacterial microorganisms that affects males because of unprotected sexual encounters with a woman who has had an abortion or miscarriage. Aim: The study aimed to investigate medicinal plants used to treat ‘u wela’ and determine their biological activity against Neisseria gonorrhoeae and Candida albicans. Setting: Eight plant species (Elaeodendron transvaalense[Burtt Davy] R.H. Archer, Albizia versicolor Welw. ex Oliv, Xanthocercis zambesiaca Baker, Cassia abbreviata subsp. beareana [Holmes] Brenan, Anthocleista grandiflora Gilg, Myrothamnus flabellifolius Welw., Mimusops zeyheri Sond, and Capparis tomentosa Lam.) used to combat ‘u wela’ were selected from the Ethnomedicinal plant’s database of over 300 medicinal plants used for medicinal purposes in humans, in the Vhembe district, Limpopo province, South Africa. Methods: The antimicrobial activity of the plant extracts was investigated against Candida albicans and Neisseria gonorrhoeae using serial dilution and bioautography assays. Results: The plant extracts of A. versicolor and C. abbreviata had excellent activity with a low minimum inhibitory concentration (MIC). value of 0.02 and 0.07 mg/mL, respectively. In bioautograms developed in benzene/ethanol/ammonia hydroxide (BEA), active compounds were visible in the extracts of A. versicolor. Conclusion: A. versicolor had excellent antimicrobial activity and may be used in traditional therapy to combat ‘u wela’. Contribution: The study has demonstrated that A. versicolor is a promising plant species that could lead to the discovery of novel drugs to combat ‘u wela’

The potential role of GLUT4 transporters and insulin receptors in the hypoglycaemic activity of Ficus lutea acetone leaf extract

BACKGROUND: Some Ficus species have been used in traditional African medicine in the treatment of diabetes. The antidiabetic potential of certain species has been confirmed in vivo but the mechanism of activity remains uncertain. The aim was to investigate the hypoglycaemic potential of ten Ficus species focussing on glucose uptake, insulin secretion and the possible mechanism of hypoglycaemic activity. METHODS: The dried and ground leaves of ten Ficus species were extracted with acetone. The dried acetone extract was reconstituted with DMSO to a concentration of 100 mg/ml which was then serially diluted and used to assay for glucose uptake in muscle, fat and liver cells, and insulin secretion in pancreatic cells. RESULTS: Only the F. lutea extract was able to modulate glucose metabolism. In comparison to insulin in the primary muscle cells, the glucose uptake ability of the extract was 33% as effective. In the hepatoma cell line, the extract was as effective as metformin in decreasing extracellular glucose concentration by approximately 20%. In the pancreatic insulin secretory assay, the extract was 4 times greater in its secretory activity than commercial glibenclamide. With F. lutea extract significantly increasing glucose uptake in the primary muscle cells, primary fat cells, C2C12 muscle and H-4-II-E liver cells, the extract may act by increasing the activity of cell surface glucose transporters. When the 3T3-L1 pre-adipocytes were compared to the primary muscle, primary fat and C2C12 cells, the differences in the former’s ability to transport glucose into the cell may be due to the absence of the GLUT4 transporter, which on activation via the insulin receptor decreases extracellular glucose concentrations. Because the pre-adipocytes failed to show any active increase in glucose uptake, the present effect has to be linked to the absence of the GLUT4 transporter. CONCLUSION: Only F. lutea possessed substantial in vitro activity related to glucose metabolism. Based on the effect produced in the various cell types, F. lutea also appears to be a partial agonist/antagonist of the insulin cell membrane receptor. While the clinical effectiveness of F. lutea is not known, this plant species does possess the ability to modify glucose metabolism

The variation in antimicrobial and antioxidant activities of acetone leaf extracts of 12 Moringa oleifera (Moringaceae) trees enables the selection of trees with additional uses

BACKGROUND : The aim of this study was to evaluate the variation in antimicrobial and antioxidant activities of the leaf acetone extracts of 12 Moringa oleifera trees harvested in order to select the best material for clonal propagation. METHODS : A two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) against a panel of fungal (Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans) and bacterial (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) species. The radical scavenging capacity was determined using 2,2 diphenyl-1-picryhydrazyl (DPPH). RESULTS : There was a large variation in antimicrobial activities with MICs between 0.04 and 2.50 mg/ml against bacteria and from 0.16 to N2.50 mg/ml against fungi. For samples harvested in winter: trees L3 and LP2 had significant activity against E. faecalis (MIC 0.08 mg/ml) and E. coli (MIC 0.04 mg/ml). Trees L5, LP1 and LP6 had weak activity against E. coli (MICs 1.25 and 2.50 mg/ml), S. aureus (MIC 1.25 mg/ml), and E. faecalis (MIC 2.50 mg/ml), while other samples had moderate activity against the four bacteria (MICs 0.16–0.63 mg/ml). From samples collected in summer: L5 (MIC 0.08 mg/ml), L6 (MIC 0.08 mg/ml after 1 h incubation), LP1 (MICs 0.08 mg/ml), LP2 (MICs 0.08 mg/ml after 1 h incubation), LP4 (0.08 mg/ml) and LP5 (MICs 0.04 and 0.08 mg/ml) had significant activity against E. faecalis (L5, L6, LP1, LP2, LP4, and LP5), S. aureus (LP1, and LP5), and E. coli (LP2, and LP5), respectively. Other extracts had weak antibacterial activity with MICs ranging from 0.16 to 0.63 mg/ml. Most of the samples harvested in winter had moderate antifungal activity: L1, L2, L3, L4, L5, L6, LP1, LP2, and LP3 had moderate activity against C. albicans (ATCC strains) with MIC of 0.63 mg/ml in all cases while L2, L3 and L4 as well as L6, LP1, LP2, LP3, LP5 and LP6 against A. fumigatus (MICs 0.63 mg/ml) and C. neoformans (MICs 0.63 mg/ml), respectively. Apart from L1 (MIC 0.31 mg/ml), L2, L3 and LP6 (MICs 0.63 mg/ml in all cases) with moderate activity, all the samples collected during summer had weak activity against A. fumigatus (MICs 1.25–2.50 mg/ml). All the extracts had a low radical scavenging activity with the IC50 values ranging from 34.72 to 109.62 μg/ml, compared to the reference standard L-ascorbic acid (IC50 2.41 μg/ml). This may be related to the extractant used. CONCLUSION : The large variation in antimicrobial activity and antioxidant activities of 24 acetone leaf extracts of 12 M. oleifera trees may lead to the selection of clonal material to serve as a source of propagation materials. Successful propagation and growth of tree LP with very good activity against E. coli and a high total activity could provide an additional use of this valuable plant species to rural people.The Limpopo Department of Agriculture (LDA), the National Research Foundation and MDA received a Postdoctoral Fellowship from the University of Pretoria. Open Access funded by SAAB.http://www.elsevier.com/locate/sajbhb2017Paraclinical Science

ANTIMYCOBACTERIAL, ANTIOXIDANT AND CYTOTOXIC ACTIVITIES OF ESSENTIAL OIL OF GALL OF PISTACIA ATLANTICA DESF. FROM ALGERIA

Background: The aim of this study was to assess the antimycobacterial, antioxidant and the cytotoxic activities of the essential oil from the gall part of Pistacia atlantica Desf from Algeria. Materials and Methods: The antimycobacterial activity was evaluated by the broth microdilution method against three species of mycobacteria: Mycobacterium smegmatis, Mycobacterium aurum and Mycobacterium fortuitum. Antioxidant activity was determined using free-radical scavenging assays. The safety of essential oil was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on C3A and Vero monkey kidney cells. Results: The minimal inhibitory concentration (MIC) values ranged from 0.16 to 2.5 mg/mL with minimal bactericidal concentration (MBC) values ranged from 0.62 to >2.5 mg/mL. The antioxidant activity showed IC50 values ranged between 417.61 - >2000µg/mL and 495.6 - >2000µg/mL for DPPH and ABTS assay respectively. The cytotoxicity assay showed LC50 ranged between 26.47 to 93.64 µg/mL against Vero cells and 74.29 to 225.40 µg/mL against C3A. The results of this study show that the essential oils from the gall of P. atlantica have low toxicity and moderate activity against fast growing M. smegmatis and M. aurum. Conclusion: These results substantiate their potential as used in the treatment of non-tuberculous mycobacterial infections. This finding is predictive of their activity against M. tuberculosis, therefore, further study might be considered to investigate the activity against pathogenic Mycobacterium strains

Antibacterial activity of crude extracts of some South African medicinal plants against multidrug resistant etiological agents of diarrhoea

Abstract Background This study evaluated the antibacterial activity of some plants used in folklore medicine to treat diarrhoea in the Eastern Cape Province, South Africa. Methods The acetone extracts of Acacia mearnsii De Wild., Aloe arborescens Mill., A. striata Haw., Cyathula uncinulata (Schrad.) Schinz, Eucomis autumnalis (Mill.) Chitt., E. comosa (Houtt.) Wehrh., Hermbstaedtia odorata (Burch. ex Moq.) T.Cooke, Hydnora africana Thunb, Hypoxis latifolia Wight, Pelargonium sidoides DC, Psidium guajava L and Schizocarphus nervosus (Burch.) van der Merwe were screened against Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, multi-resistant Salmonella enterica serovar Isangi, S. typhi, S. enterica serovar Typhimurium, Shigella flexneri type 1b and Sh. sonnei phase II. A qualitative phytochemical screening of the plants extracts was by thin layer chromatography. Plants extracts were screened for antibacterial activity using serial dilution microplate technique and bioautography. Results The TLC fingerprint indicated the presence of terpenoids and flavonoids in the herbs. Most of the tested organisms were sensitive to the crude acetone extracts with minimum inhibitory concentration (MIC) values ranging from 0.018–2.5 mg/mℓ. Extracts of A. striata, C. uncinulata, E. autumnalis and P. guajava were more active against enteropathogens. S. aureus and Sh. flexneri were the most sensitive isolates to the crude extracts but of significance is the antibacterial activity of A. arborescens and P. guajava against a confirmed extended spectrum betalactamase positive S. enterica serovar Typhimurium. Conclusion The presence of bioactive compounds and the antibacterial activity of some of the selected herbs against multidrug resistant enteric agents corroborate assertions by traditional healers on their efficacies