Characterization of antifungal compounds isolated from Combretum and Terminalia species (Combretaceae)

Several investigations into the antimicrobial activity of members of the Combretaceae have been undertaken in recent years. Although the antibacterial properties of various species of Combretum, Terminalia and Pteleopsis have been investigated in depth, this is not the case for their antifungal properties. Due to the increasing importance of fungal infections the aim is to address this by focusing on antifungal activities of Combretaceae species. This was done by focusing on the following objectives: Developing minimum inhibitory concentration (MIC) and bioautography procedures for fungi to be used in the laboratory in order to screen Combretum andTerminalia species for antifungal activity. Selecting three or four species for further investigation based on antifungal activity and availability. Isolating the antifungal compounds from one or more of the selected species. Determining the chemical structure and in vitro biological activity of the antifungal compound. Developing and applying a protocol and determining in vivo antifungal activity of Combretum and Terminalia extracts and isolated compounds in rats infected with different fungal pathogens. Leaves of 24 Combretum and 6 Terminaliaspecies were collected in the Lowveld National Botanical Gardens (LNBG) in Nelspruit. After the dried plants were milled to a fine powder, they were extracted with hexane, dichloromethane, acetone and methanol. Chemical constituents of the 120 extracts were analyzed by thin layer chromatography (TLC). The TLC plates were developed with one of the three eluent systems developed in our laboratory that separate components of Combretaceae extracts well i.e.: Ethyl acetate/methanol/water (40:5.4:5) [EMW] (polar/neutral), Chloroform/ethyl acetate/formic acid (5:4:1) [CEF] (intermediate polarity/acidic) and Benzene/ethanol/ammonia hydroxide (90:10:1) [BEA] (non-polar/basic). To detect the separated compounds, vanillin-sulphuric acid-methanol was sprayed on the chromatograms and heated at 110 °C to optimal colour development. Methanol was the best extractant, extracting a greater quantity of plant material than any of the other solvents. There was similarity in the chemical composition of the non-polar compounds of extracts using extractants of varying polarity Qualitative analysis of antioxidant activity, the 2, 2,diphenyl-1-picrylhydrazyl (DPPH) assay on TLC plates was used as a screen test for the radical scavenging ability of the compounds present in the different 120 extracts. TLC-DPPH screening method indicated the presence of antioxidant compounds in some of the extracts tested, with C. woodii and C. hereroense showing the most prominent antioxidant activity. Methanol and acetone extracted the most antioxidant compounds based on DPPH TLC. In vitro studies coupled with the phytochemical analysis confirm that the extracts had antioxidant activity. The solvent tolerance of the microorganisms was tested using the following solvents; DMSO, acetone, methanol and ethanol. In order to determine the maximum concentration at which different solvents would allow the test microorganisms to reach normal growth, different concentrations from 10 to 100% were used. Uninhibited growth was evaluated as no toxic effects of the solvent. Methanol and ethanol were found to be toxic. The growths of the fungi were not affected by DMSO and acetone concentrations up to 60%. A serial microdilution assay was used to determine the minimum inhibitory concentration (MIC) values for plant extracts using tetrazolium violet reduction as an indicator of growth. This method had previously been used only for antibacterial activities. To apply it to measuring antifungal activities, a slight modification was made to suit fungal growth conditions. The following fungal pathogens were used: yeasts (Candida albicans and Cryptococcus neoformans), thermally dimorphic fungi (Sporothrix schenckii) and moulds (Aspergillus fumigatus and Microsporum canis). To determine MIC values, growth was checked after 24 and 48 hours to determine the end point. The MIC values of most of the extracts were in the order of 0.08 mg/ml and some had values as low as 0.02 – 0.04 mg/ml after 24 hours incubation. TLC plates were loaded with 100 ㎍ (5 ㎕ of 20 mg/ml) of each of the extracts. The prepared plates were developed in the three different mobile systems used: CEF, BEA and EMW. The chromatograms were dried for a week at room temperature under a stream of air to remove the remaining solvent. The TLC plates developed were inoculated with a fine spray of the concentrated suspension containing approximately 109 organisms per ml of actively growing fungi e.g. conidia for A. fumigatus and yeast cells (blastocysts) for the other fungi in a Biosafety Class II cabinet (Labotec, SA) cupboard. The plates were sprayed until they were just wet, and after drying were sprayed with a 2 mg/ml solution of INT. White areas indicate where reduction of INT to the coloured formazan did not take place due to the presence of compounds that inhibited the growth of tested fungi. During this study we experienced a number of difficulties. Firstly I found that preparing cultures some days before spraying them makes it difficult to get good results, possibly due to quick mycelial overgrowth and blockage of the spray gun with mycelia. The new method was developed. This procedure led to reduced overgrowth of the mycelia. In the study of biologically active compounds from extracts, it was indicated that the extracts had antifungal compounds. Fractionation and bioassay-guided isolation of the antifungal compounds was undertaken on the crude extracts of C. nelsonii, based on very low MIC’s of the crude extracts on all tested pathogens, it had several compound which are active against all pathogens, lastly it is one of the Combretum species which have never being worked on. Antifungal compound was successfully isolated from the leaves of C. nelsonii. The structure was elucidated. After structure elucidation bioassays of isolated active compounds was done to confirm that the compound isolated is the one expected, and how active the compound is, on its own. The compound was very active against all tested pathogens. Cytotoxicity of the acetone extracts of C. imberbe, C. nelsonii, C. albopunctactum and T. sericea were evaluated using Brine shrimp (Artemia salina assay and tetrazolium-based colorimetric assay (MTT assay) on Vero monkey kidney cells. These four extracts were chosen because of the good in vitro antifungal activity of crude extracts and there was intention of using them in in vivo studies in animal models. The results on brine shrimps indicated that the four leaf extracts have LC50 values above 20 ㎍/ml, the recommended cut-off point for detecting cytotoxic activity. Using MTT assay it was found that the four extracts did not suppress mitochondrial respiration in monkey kidney cells. Only C. imberbe was closer to the cut-off value (200 µg/ml), which was used by other authors. In searching for cytotoxic activity to the criteria of the American National Cancer Institute, the LC50 limit to consider a crude extract promising for further purification is lower than 30 µg/ml. In vivo antifungal activity was investigated on the wound irritancy and efficacy of the four most promising, Combretum nelsonii, Combretum imberbe, Combretum albopunctactum and Terminalia sericea extracts applied topically to skin wounds in fungal infected skin wound of rat model. Wound irritancy and wound healing were evaluated by macroscopical, physical and histological methods. Aspects evaluated include wound healing, erythema, exudate formation and possible toxic effects of the extracts. Twenty rats were used in two pilot studies (Exploratory studies and Infection with different pathogens). During the pilot studies rats were not irritated by treatment of infection. The wound healed within three weeks. Only one rat was terminated due to weight loss and it was found that nasal discharge was due to external factors, which were not related to the experiment. The clinical treatment of skin infected with pathogens continues to be a major problem especially in immuno-compromised patients. Therapeutic agents selected for the treatment of infected wounds had ideally shown antifungal activity on in vitro studies. I investigated whether these agents would improve phases of wound healing without producing deleterious side effects. All the parameters showed that the crude extracts and amphotericin B were effective in decreasing formation of the exudate, increasing crust formation and that they have antifungal activities used in in vivo studies. Acetone extract of leaves of C. nelsonii, C. albopunctactum, C. imberbe and T. sericea possessed remarkable growth inhibitory activities against fungal pathogens. Acetone extracts of leaves and isolated compound demonstrated wound healing properties comparable with that of antibiotic powder (amphotericin B). The results of this study in general indicate that the Terminalia and Combretum species possess substantial antifungal properties. This explains the use of these plants in folk medicine for the treatment of various diseases related to fungal infections.Thesis (PhD (Phytomedicine))--University of Pretoria, 2006.Paraclinical Sciencesunrestricte

ANTIOXIDANT AND ANTIBACTERIAL PROPERTIES OF ZIZIPHUS MUCRONATA AND RICINUS COMMUNIS LEAVES EXTRACTS

Background: Plants have always been a successful source of remedy from nature. With the widespread use of medicinal plants by indigenous people, the search for biologically active agents is relevant as these plants have the potential to provide pharmacologically active compounds. This study aimed for investigating the effect of antioxidant and antibacterial properties of Ziziphus mucronata Willd (Rhamnaceae) and Ricinus communis L. (Euphorbiaceae). Materials and methods: Antibacterial activity was evaluated using microdilution assay and bioautography. Antioxidant activities were determined by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH). In vitro cytotoxicity was determined using the tetrazolium-based colorimetric assay. Results: R. communis leaves had eight secondary metabolites. Quantitative assay for R. communis, chloroform and methanol extracts had very high antioxidant activity compared to vitamin C. Plants extracts from all solvents exhibited high antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa with minimum inhibitory concentration (MIC) values between 0.21 and 1.05 mg/ml. Most of the antibacterial compounds observed on bioautography had Rf values ranging from 0.21 to 0.88. Z. mucronata had LC50 of 105.5 μg/ml and R. communis 131.8 μg/ml on Vero cells. Conclusion: This study revealed that both plants had free radical scavenging and antibacterial activities

Anti-mycobacteria potential and synergistic effects of combined crude extracts of selected medicinal plants used by Bapedi traditional healers to treat tuberculosis related symptoms in Limpopo Province, South Africa

BACKGROUND : Tuberculosis is an infectious communicable disease and the causative agent of the disease has over the years developed resistance to streamline chemotherapeutic agents with dire consequences and there is a need for development of new and more potent alternatives. METHODS : Constituents of leaves material of Combretum heroroense, Citrus lemon and Apodytes dimidiata were serially extracted using solvents of varying polarity. TLC finger print profile of the different extracts were determined by spraying eluted plates with vanillin sulphuric acid and 2, 2- diphenylpicryl hydrazyl (DPPH) for the presence of antioxidant constituents. Presence of different phytochemicals was determined using standard chemical test. Bioautography was used to determine the number of compounds present in sub-fractions active against Mycobacterium smegmatis. Minimum inhibitory concentration (MIC) values extract and sub-fractions were determined using serial microplate dilution method against M. smegmatis (ATCC 1441), M. tuberculosis (ATCC H37Rv) and multi-drug resistant TB (MDR-TB) field strain. Synergy of the crude extracts of the three plants was determined using microplate dilution method against M. smegmatis. RESULTS : Mass extracted by different solvents was less than 6% dry weight for all the plants. Phlobatannins were not detected in A. dimidiata, C. heroroense and C. lemon as well as cardiac glycosides in C. lemon and A. dimidiata, and saponins in C. heroroense. Sub-fractions of the different plants were shown to contain constituents with antioxidant activity with the highest number detected in C. heroroense. Bioautography results reveal the presence of a compound(s) in the ethyle acetate sub-fraction of C. heroroense and butanol, methanol/water, ethyl acetate and water no.2 subfractions of A. dimidiata, active against M. smegmatis that were not shown to have antioxidant capacity. MIC results for different crude extracts of the three plants against M. smegmatis ranges from 0.1 to 3 mg/ml. The average MIC for the synergistic effect of the plants ranged from 0.04 mg/ml to 1.25 mg/ml. An activity greater than that obtained for the reference drugs was shown for the butanol and hexane fractions of A. dimidiata (0.47 mg/ml) against the field strain of MDR-TB while that obtained for the M.TB (ATCC H37Rv) was 0.31 mg/ml. CONCLUSION : A significant finding shown in this study reveals the potent anti-mycobacteria potential of sub-fractions of A. dimidiata against MDR-TB field strain that can lead to the isolation of compounds that can be used to counter resistant strains of tuberculosis.The NRF (Reference: IFR1203260814; Grant No: 81341 and University of Limpopo (Grant no: R800).http://www.biomedcentral.com/bmccom/plementalternmedam2017Paraclinical Science

ANTI-BACTERIAL AND ANTI-OXIDANT ACTIVITIES OF LEAF EXTRACTS OF COMBRETUM VENDAE (COMBRETECACEA) AND THE ISOLATION OF AN ANTI-BACTERIAL COMPOUND.

Background: Combretum vendae A.E. van Wyk (Combretaceae) is used for the treatment of bacterial related infections and oxidative related diseases by indigenous people of South Africa. Dried leaves extracts of C. vendae were investigated for bioactivity against a variety of bacterial strains and their antioxidant potential evaluated. Materials and methods: Constituents of leaf material were serially extracted using solvents of varying polarities, TLC chromatograms of the fractions were sprayed with 2,2 diphenyl-1-picrylhydrazyl (DPPH) to determine the presence of antioxidant compounds. Bio-autography was used to determine the number of antibacterial compounds active against Staphylococcus aureus, Enterococcus faecalis, Eschericha coli and Pseudomonas aeruginosa. Minimum inhibitory concentration (MIC) values were determined using serial microplate dilution method. The chloroform fraction was subjected to bio-assay guided column chromatography to isolate the active compound. Results: The mass extracted by different solvents was below 10% dry weight. MIC values for different extracts against different pathogens ranges from 0.08 to 0.64 mg/ml. The compound isolated was identified as acacetin having an Rf value of 0.28 following elution in the Ethanol: Methanol: Water [E: M: W (10: 1.35: 1 v/v). Acacetin had MIC values ranging from 0.16 to 0.35 mg/ml. Conclusion: We report for the first time the isolation of acacetin as the main antibacterial compound from the leaves of Combretum vendae

Isolation and chemical structural characterisation of a compound with antioxidant activity from the roots of Senna italica

Senna italica, a member of the Fabaceae family (subfamily Caesalpiniaceae), is widely used in South African traditional medicine to treat a number of disease conditions. Aqueous extracts of the plant are mainly used to treat sexually transmitted infections and intestinal complications. The roots of S. italica were ground to a fine powder and sequentially extracted with n-hexane, dichloromethane, acetone, and methanol using serial exhaustive extraction (SEE) method. Thin layer chromatography was used to analyse the phytochemical composition of the extracts and DPPH radical scavenging method to detect the presence of antioxidant compounds. The bioassay guided fractionation of the acetone fraction afforded an antioxidant compound with free radical scavenging activity.The isolated compoundwas subsequently identified as 3,4????,5-trihydroxystilbene (resveratrol).This study represents the first report of the stilbene resveratrol in S. italica.The National Research Foundation (NRF) South Africa (Gun no. N710), the Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, and Phytomedicine Programme, University of Pretoria.http://www.hindawi.com/journals/ecam/am2013mn201

In Vitro

Ricinus communis has been utilized traditionally as medicine to treat inflammatory related diseases including wounds, sores, and boils. The leaves of R. communis were sequentially extracted with n-hexane, dichloromethane, acetone, and methanol using serial exhaustive extraction method. Antioxidant activity of all crude extracts was quantitatively measured against 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) free radical molecules using ABTS+ assay. Cytotoxic effect and anti-inflammatory activity of R. communis leaves extracts were evaluated on Human Caucasian skin fibroblast and Raw 264.7 macrophage cell lines, respectively. Methanol extract had the highest percentage free radical (ABTS+) scavenging activity of 95% at 2.50 mg/mL, acetone 91%, dichloromethane 62%, and hexane the least (50%). Percentage scavenging activity of ABTS+ free radical molecules increases with increase in concentrations of the plant extracts. Hexane and dichloromethane extracts had more than 90% cell viability at 100 µg/mL after 24 and 48 hours of exposure. Methanol extract had LC50 of 784 µg/mL after 24-hour exposure, hexane had 629.3 µg/mL and dichloromethane 573.6 µg/mL, and 544.6 µg/mL was the lowest with acetone extract. The study present the first report on the scavenging activity of R. communis leaf extracts against ABTS+ radicals and cytotoxic effects on human Caucasian skin fibroblast cell lines

Validation of Antimycobacterial Plants Used by Traditional Healers in Three Districts of the Limpopo Province (South Africa)

The aim of the study was to scientifically evaluate the antimycobacterial activity of selected indigenous medicinal plants from the Limpopo Province used for the treatment of humans with symptoms of Mycobacterium tuberculosis. The leaves of five plant species (Apodytes dimidiata, Artemisia, Combretum hereroense, Lippia javanica, and Zanthoxylum capense) were collected from the Lowveld National Botanical Garden in Nelspruit, South Africa. The dried leaves were powdered and extracted using hexane, dichloromethane, acetone, and methanol. Antimycobacterial activity was evaluated using microdilution assay and bioautography and -iodonitrotetrazolium violet (INT) as indicator. Antioxidant activities were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH). Phytochemical content of extracts was further evaluated. The acetone extracts of L. javanica displayed antioxidant activity on BEA chromatogram. T Acetone extracts of A. afra had MIC value of 0.39 mg/mL against Mycobacterium smegmatis ATCC 1441. Acetone extracts of C. hereroense and L. javanica had MIC value of 0.47 mg/mL. Four bands that inhibited the growth of M. smegmatis were observed at values of 0.12, 0.63, and 0.87 on BEA and 0.73 on EMW. The plant species A. dimidiata, A. afra, C. hereroense, and L. javanica in this study demonstrated their potential as sources of anti-TB drug leads

Validation of Antimycobacterial Plants Used by Traditional Healers in Three Districts of the Limpopo Province (South Africa)

The aim of the study was to scientifically evaluate the antimycobacterial activity of selected indigenous medicinal plants from the Limpopo Province used for the treatment of humans with symptoms of Mycobacterium tuberculosis. The leaves of five plant species (Apodytes dimidiata, Artemisia, Combretum hereroense, Lippia javanica, and Zanthoxylum capense) were collected from the Lowveld National Botanical Garden in Nelspruit, South Africa. The dried leaves were powdered and extracted using hexane, dichloromethane, acetone, and methanol. Antimycobacterial activity was evaluated using microdilution assay and bioautography and ρ-iodonitrotetrazolium violet (INT) as indicator. Antioxidant activities were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH). Phytochemical content of extracts was further evaluated. The acetone extracts of L. javanica displayed antioxidant activity on BEA chromatogram. T Acetone extracts of A. afra had MIC value of 0.39 mg/mL against Mycobacterium smegmatis ATCC 1441. Acetone extracts of C. hereroense and L. javanica had MIC value of 0.47 mg/mL. Four bands that inhibited the growth of M. smegmatis were observed at Rf values of 0.12, 0.63, and 0.87 on BEA and 0.73 on EMW. The plant species A. dimidiata, A. afra, C. hereroense, and L. javanica in this study demonstrated their potential as sources of anti-TB drug leads

Screening of twenty-four South African Combretum and six Terminalia species (Combretaceae) for antioxidant activities

The dried leaves of Combretum and Terminalia species (Combretaceae) were extracted with acetone, hexane, dichloromethane and methanol. Thin layer chromatography (TLC) plates were developed under saturated conditions and sprayed with 0.2% 2,2-diphenyl-1-picryl hydrazyl (DPPH) in methanol for antioxidant screening. Visualization of separated bands exhibiting antioxidant activities enabled the localization and the subsequent identification of the potential active compounds. The acetone and methanol extracts displayed the presence of antioxidant activity after spraying the chromatogram with DPPH. Hexane and dichloromethane extracts did not have any antioxidant activity. C. hereroense had the highest number of active compounds, followed by C. collinum ssp. taborense, which were 16 and 10, respectively. Acetone extracts of all tested Combretum species had 53 active bands and methanol had 55. All Terminalia species extracted with acetone and methanol had antioxidant activity. T. gazensis and T. mollis methanol extracts had 11 and 14 active compounds respectively in one of the solvent systems used. The qualitative DPPH assay on TLC was successfully used in this study to systematically assess the total antioxidant activity of the Combretum and Terminalia species extracts