Quantifying the bioactivity of plant extracts during screening and bioassay-guided fractionation

No Abstracthttp://www.elsevier.de/phymedhb2014mn201

A proposal towards a rational classification of the antimicrobial activity of acetone tree leaf extracts in a search for new antimicrobials

Many scientists investigate the potential of finding new antibiotics from plants, leading to more than a thousand publications per year. Many different minimum inhibitory concentrations of extracts have been proposed to decide if an extract has interesting activity that could lead to the discovery of a new antibiotic. To date, no rational explanation has been given for the selection criteria different authors have used. The cumulative percentage of plant extracts with different activities from a large experiment determining the activity of 714 acetone tree leaf extracts of 537 different South African tree species against 4 nosocomial pathogenic bacteria and 2 yeasts was calculated using a widely accepted serial dilution microplate method with p-iodonitrotetrazolium violet as indicator of growth. All the extracts were active at a concentration of 2.5 mg/mL. The formula, % of active extracts = 439 × minimum inhibitory concentration in mg/mL1.5385, describes the results for minimum inhibitory concentrations below 0.16 mg/mL, with a correlation coefficient of 0.9998. A rational approach could be to determine the minimum inhibitory concentrations of the most active 1, 3, 9, 25, 50, and > 50% of a large number of plant extracts investigated against these six important microbial pathogens. Starting with an extract concentration of 10 mg/mL, I propose the following classification based on minimum inhibitory concentrations: outstanding activity < 0.02 mg/mL, excellent activity 0.021 – 0.04 mg/mL, very good activity 041 – 0.08 mg/mL, good activity 0.081 – 0.16 mg/mL, average activity 0.161 – 0.32 mg/mL, and weak activity > 0.32 mg/mL. Higher minimum inhibitory concentrations may still be effective in ethnopharmacological studies.Many scientists investigate the potential of finding new antibiotics from plants, leading to more than a thousand publications per year. Many different minimum inhibitory concentrations of extracts have been proposed to decide if an extract has interesting activity that could lead to the discovery of a new antibiotic. To date, no rational explanation has been given for the selection criteria different authors have used. The cumulative percentage of plant extracts with different activities from a large experiment determining the activity of 714 acetone tree leaf extracts of 537 different South African tree species against 4 nosocomial pathogenic bacteria and 2 yeasts was calculated using a widely accepted serial dilution microplate method with p-iodonitrotetrazolium violet as indicator of growth. All the extracts were active at a concentration of 2.5 mg/mL. The formula, % of active extracts = 439 × minimum inhibitory concentration in mg/mL1.5385, describes the results for minimum inhibitory concentrations below 0.16 mg/mL, with a correlation coefficient of 0.9998. A rational approach could be to determine the minimum inhibitory concentrations of the most active 1, 3, 9, 25, 50, and > 50% of a large number of plant extracts investigated against these six important microbial pathogens. Starting with an extract concentration of 10 mg/mL, I propose the following classification based on minimum inhibitory concentrations: outstanding activity < 0.02 mg/mL, excellent activity 0.021 – 0.04 mg/mL, very good activity 041 – 0.08 mg/mL, good activity 0.081 – 0.16 mg/mL, average activity 0.161 – 0.32 mg/mL, and weak activity > 0.32 mg/mL. Higher minimum inhibitory concentrations may still be effective in ethnopharmacological studies.The National Research Foundation, South Africa Incentive funding.https://www.thieme-connect.com/products/ejournals/journal/10.1055/s-00000058hj2022Paraclinical Science

Ovicidal and larvicidal activity of Cassia alata leaf acetone extract and fractions on Haemonchus contortus : in vitro studies

CONTEXT : The failure of modern anthelmintics to control nematode parasites of sheep and goats is a reality on many farms in the tropical/subtropical regions of the world. This necessitates chemotherapeutic control alternatives and plant secondary metabolite with activity is one of those potential solutions. OBJECTIVE : This study was design to evaluate the efficacy of solvent: solvent fractions of Cassia alata Gelenggang Besar (Leguminosae) leaf acetone extract against Heamonchus contortus Rudolphi (Trichostrongylidae). MATERIALS AND METHODS : C. alata leaf was extracted with 70% acetone and fractions were obtained by solvent: solvent group separation procedures. The acetone extract and the fractions were tested by egg hatch assay (EHA) and larval development and viability assay to assess relative bioactivity against H. contortus eggs and larvae. RESULTS : The extracts inhibited egg hatchability and killed infective larvae of H. contortus in a concentration-dependent manner. The best-fit LC(50) values were 0.562, 0.243, 0.490, 0.314, and 0.119 mg/mL for the acetone extract, chloroform, hexane, butanol and 35% water in methanol fractions, respectively, when tested against nematode eggs. The best-fit LC(50) values were 0.191, 0.505, 1.444, 0.306, and 0.040 mg/mL for acetone extract, chloroform, hexane, butanol and 35% water in methanol fractions, respectively, when tested against larvae. The 35% water in methanol fraction was the most active against the larvae and eggs of H. contortus demonstrating the lowest LC(50) values DISCUSSION AND CONCLUSION : This study demonstrates that the leaf extracts of C. alata have anthelmintic activity; therefore it could find application in the control of helminths in livestockThe National Research Foundation South Africahttp://www.tandfonline.com/loi/iphb20hb2017Paraclinical Science

The South African floricultural industry and the Plant Breeders' Rights Act : a short review

The floriculture industry has been identified as one of the industries with an opportunity to grow and contribute positively to the economy of South Africa. The largest number of plant breeders’ rights applications filed in South Africa is for ornamental plants. This article gives a short review of the South African floricultural industry, some features of the Plant Breeders’ Rights Act, 1976 (Act No. 15 of 1976), and the trends in plant breeders’ rights applications and grants for ornamental plants over the past decade.http://www.elsevier.com/locate/worpatinhb2017Paraclinical Science

Anti-inflammatory, anticholinesterase and antioxidant activity of leaf extracts of twelve plants used traditionally to alleviate pain and inflammation in South Africa

ETHNOPHARMAOLOGICAL RELEVANCE :Oxidative stress and inflammatory conditions are among the pathological features associated with the central nervous system in Alzheimer’s disease. Traditionally, medicinal plants have been used to alleviate inflammation, pains and also other symptoms possibly associated with Alzheimer’s disease. Therefore, the present study was designed to determine the in vitro antiinflammatory, antioxidant and anticholinesterase activity of twelve South African medicinal plants traditionally used to alleviate pain and inflammation. MATERIALS AND METHODS : Nitric oxide (NO) production in LPS-activated RAW 264.7 macrophages and 15-lipoxygenase (LOX) inhibitory assay were used to evaluate the anti-inflammatory activity. Acetylcholinesterase inhibition was assessed by using a modification of the Ellman’s method. Antioxidant activity, total phenolic and total flavonoids contents were determined using standard in vitro methods. RESULTS : The extract of Burkea africana had the highest anti-15-lipoxygenase activity with 85.92% inhibition at 100 μg/mL. All the extracts tested inhibited nitric oxide (NO) production in a dose dependant manner in LPS-stimulated RAW 264.7 macrophages. However, extracts from Leucaena leucocephala, Lippia javanica inhibited the production of NO by 97% at a concentration of 25 μg/mL. In addition, both Leucaena leucocephala and Englerophytum magaliesmontanum had strong activity against acetylcholinesterase with IC50 values of 118 μg/mL and 160 μg/mL respectively. Hight levels of phenolics and flavonoids were found in Leucaena leucocephala, Lippia javanica and Burkea africana. The correlation with antioxidant activities was not strong indicating that other metabolites may also be involved in antioxidant activity. CONCLUSIONS : The results obtained in this study validate the use of leaf extracts of these plants in South African traditional medicine against inflammation. Extracts of these plants species might be of value in the management of various diseases emerging from oxidative stress and related degenerative disorders.National Research Foundation (NRF) and Medical Research Council (MRC).http://www.elsevier.com/locate/jethpharm2016-02-28hb201

Which tree orders in southern Africa have the highest antimicrobial activity and selectivity against bacterial and fungal pathogens of animals?

BACKGROUND: The study randomly screened leaf extracts of several hundred southern African tree species against important microbial pathogens to determine which taxa have the highest activity and may yield useful products to treat infections in the animal health market. METHODS: We determined the antibacterial and antifungal activity of 714 acetone leaf extracts of 537 different tree species against Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Cryptococcus neoformans. A sensitive serial dilution microplate method was used. RESULTS: Several extracts had MICs as low as 0.02 mg/ml. We analysed 14 out of the 38 tree orders where we determined the activity of more than 8 different tree species representing 89% of all species examined. There were statistically significant differences in some cases. Celastrales, Rosales and Myrtales had the highest activity against Gram-positive bacteria, the Myrtales and Fabales against the Gram-negative bacteria and the Malvales and Proteales against the fungi. Species present in the Asterales followed by the Gentiales and Lamiales had the lowest activities against all the microorganisms tested. Fabales species had the highest activities against all the microorganisms tested. There was substantial selectivity in some orders. Proteales species had very high activity against the fungi but very low activity against the bacteria. The species in the Celastrales and Rosales had very low antifungal activity, low activity against Gram-negative bacteria and very high activity against Gram-positive bacteria. CONCLUSION: Against all classes of microorganisms, the four orders containing species with the highest average antimicrobial activities also contained several species with low activities against different pathogens and vice versa. These results therefore should be used with circumspection in selecting tree orders that would yield the highest probability of finding species with promising activities. Nevertheless there was a twofold increase in probability of finding extracts with interesting antifungal activity from orders with high mean activity than from orders with low mean activity. The probability increased to threefold and fivefold for Gram-positive and Gram-negative bacteria respectively.The National Research Foundation (NRF)http://www.biomedcentral.com/bmccomplementalternmedam201

In vitro antibacterial activity of seven plants used traditionally to treat wound myiasis in animals in southern Africa

In the extreme situation of subsistence farming where insecticides and other veterinary medicines are either unavailable or unaffordable, the use of plants in the treatment of wound myiasis in livestock has been reported worldwide. However, the exact effect of these plants on myiatic wounds has not been established. This study was therefore undertaken to establish the biological activity of seven species of plants which are used traditionally and are claimed to be effective in the treatment of wound myiasis. Plants that have a wide distribution in southern Africa were selected. This paper focuses on the antibacterial activity of these plants on bacteria known to be among the common contaminants of wounds. It has been shown that bacterial action on wounds produce compounds which have an odour that serve as an attractant of myiasis-causing flies. The antibacterial activity of the plants was investigated using a microdilution assay and bioautography methods. All the tested plants had inhibitory activity against the test bacteria. Inhibiting bacterial activity reduces the attractants of myiasis-causing flies to the wound. Thus, inhibiting bacteria action on wounds will interfere with the development of wound myiasis. This could be one of the mechanism through which the plants that are used traditionally in the treatment of wound myiasis work.The University of Pretoria and the National Research Foundation. L. Mukandiwa gratefully acknowledges the financial support from German Academic Exchange Service, DAAD, during the period of this study.http://www.academicjournals.org/JMPRam2013mn201

Repellent and mosquitocidal effects of leaf extracts of Clausena anisata against the Aedes aegypti mosquito (Diptera : Culicidae)

Mosquitoes are rapidly developing resistance to insecticides that millions of people relied on to protect themselves from the diseases they carry, thereby creating a need to develop new insecticides. Clausena anisata is used traditionally as an insect repellent by various communities in Africa and Asia. For this study, the repellency and adulticidal activities of leaf extracts and compounds isolated from this plant species were evaluated against the yellow fever mosquito, Aedes aegypti. In the topical application assays, using total bites as an indicator, repellency was dose dependent, with the acetone crude extract (15 %) having 93 % repellence and the hexane fraction (7.5 %) 67 % repellence after 3 h. Fractionation resulted in a loss of total repellence. As mosquito-net treating agents, the acetone and hexane extracts of C. anisata, both at 15 %, had average repellences of 46.89 ± 2.95 and 50.13 ± 2.02 %, respectively, 3 h after exposure. The C. anisata acetone extract and its hexane fraction caused mosquito knockdown and eventually death when nebulised into the testing chamber, with an EC50 of 78.9 mg/ml (7.89 %) and 71.6 mg/ml (7.16 %) in the first 15 min after spraying. C. anisata leaf extracts have potential to be included in protection products against mosquitoes due to the repellent and cidal compounds contained therein.The University of Pretoria and the National Research Foundation.http://link.springer.com/journal/113562017-06-30hb2016Paraclinical Science

Larvicidal activity of leaf extracts and seselin from Clausena anisata (Rutaceae) against Aedes aegypti

The Aedes aegypti mosquito is a vector of various diseases in both humans and livestock.Mosquito control focuses on reducing the longevity as well as the population of mosquitoes to lessen their damage on human and animal health. It entails several strategies such as environmental management, insecticide treatments, and molecular entomological approaches. Environmental management centres on elimination of breeding sites, however mosquitoes can breed in sites that cannot be eliminated. Resultantly, focus is turned onto mosquito larvae control. The objective of this study was to evaluate the larvicidal activity of extracts and compounds from Clausena anisata against A. aegypti. The World Health Organization guidelines for testing of mosquito larvicides were used. The acetone, dichloromethane and hexane crude leaf extracts were evaluated in a preliminary screening for larvicidal activity at the concentrations of 12.5, 25, 50, 100 and 200 ppm. Batches of 25 third-instar larvae were transferred into cups each containing test solutions and larval mortality was recorded 24 h and 48 h after exposure. Acetone was used as the solvent control whilst permethrin was used as a positive control. Only the n-hexane extract caused mortality at the tested concentrations, thus it was further tested at 40, 60, 80, 100, and 120 ppm and had LC50 values of 68.30 and 59.65 ppm after 24 h and 48 h respectively. A stored hexane extract, of 2months,was also evaluated under simulated field conditions to establish stability of extract. It caused about 90% mortality when tested at 100 ppm. The n-hexane extract was subjected to open column chromatography on silica gel to isolate the active compound. The isolated compound was identified as the pyranocoumarin, seselin. Dose dependentmortality was observed in the larvae exposed to seselin. The LC50 values at 24 and 48 h were 13.90 and 9.96 ppmrespectively. Results obtained from this study indicate a potential of the incorporation of C. anisata extracts into the control of mosquito populations.University of Pretoria (UP Postdoctoral Fellowship Fund) and the National Research Foundation (Grant number: Eloff 95991).http://www.elsevier.com/locate/sajb2016-09-30hb201

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 EC50 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 EC50 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 LC50 of 0.17 mg/ml, while Cyathea dregei was the most toxic plant with an LC50 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.MA participated in the design of the study, carried out field work, prepared the extracts, participated in all assay, analysed the data and wrote first draft and subsequent drafts of the manuscript. VN participated in the design and coordination of the study, supervised the study, analysed the data and revised the draft manuscript. JNE participated in the design and coordination of the study, supervised the study and revised the manuscript. All authors read and approved the final manuscript.The National Research Foundation and University of Pretoria provided funds, the Pretoria National Botanic gardens allowed us to collect plant material. The study of one of us (MA) was made possible by the University of Agriculture Makurdi and Tertiary Education Tax Fund (TETFund), Nigeria.http://www.biomedcentral.com/1746-6148/9/38am2013mn201