Isolation and characterization of compounds from Podocarpus henkelii (Podocarpaceae) with activity against bacterial, fungal and viral pathogens

Diseases caused by bacteria, fungi and viruses pose a significant threat especially to poor rural communities. Viral infections are frequently complicated by secondary bacterial and fungal infections which remain a major challenge globally and in particular, in sub Sahara Africa amongst humans and animals alike. The main aim of this study was to develop a low toxicity plant extract or isolated compound active against viral, bacteria and fungal pathogens from selected plant species. Seven tree species that were investigated were Acokanthera schimperi, Carissa edulis, Ekebergia capensis, Podocarpus henkellii, Plumbago zeylanica, Annona senegalensis and Schrebera alata traditionally used in the treatments of various ailments were selected and extracted using solvents of varying polarity. Extracts of selected plants were tested for activity against two Gram positive and two Gram negative bacterial namely Enterococcus faecalis and Staphylococcus aureus and two Gram-negative species, Pseudomonas aeruginosa and Escherichia coli respectively, three fungal pathogens: Candida albicans, Cryptococcus neoformans and Aspergillus fumigates and four enveloped animal viruses: feline herpes virus–1 (FHV-1, dsDNA), canine distemper virus (CDV, ssRNA), canine parainfluenza virus-2 (CPIV-2, ssRNA) and lumpy skin disease virus strain V248/93 (LSDV, dsDNA). The presence of antioxidant constituents in the different extracts and cytotoxicity against three cell types CRFK, bovine dermis and Vero cells were determined. Bioautography and the serial microplate dilution methods were used to determine the number of antimicrobial compounds and antimicrobial activity of extracts against bacterial and fungal pathogens. Virucidal and attachments assays were used to determine the activity against viral pathogens. Qualitative antioxidant activities of extracts were tested using the DPPH reagent and cytotoxicity using the MTT assay. Biological activity was observed in all the extracts against one or more organisms on bioautography. The intermediately polar system (CEF) separated more active constituents. Some extracts had compounds with similar Rf values active against one or more organisms. In both the antibacterial and antifungal assays, acetone extracts had the highest activity followed by DCM against one or more pathogens. Hexanes extracts were the least active. P. henkellii extracts had more active compounds against the bacteria and Annona senegalensisagainst the fungi. In the micro-dilution assay, S. aureus was the most susceptible bacterial organism to extracts of the different plant, followed by P. aeruginosa andEscherichia coli, and E. faecalis the least. C. neoformans on the other hand was the most susceptible fungal pathogen. In the antiviral assay, although activity was observed with hexane extracts of some plants in the virucidal assay, the most potent inhibition was observed with the acetone and methanol extracts of Podocarpus henkelii against CDV and LSDV in the virucidal assay and acetone extracts in the attachment assay. In general the hexane was the least toxic while the intermediate polarity extracts were generally the most toxic indicating that highly polar compounds were possibly poorly or highly absorbed through membranes in the former and later respectively. Of the three cell types used CRFK was the most sensitive followed by bovine dermis and Vero cells the least. Cytotoxicity studies of extracts of the different plants revealed A. senegalensis and A. schimperi extracts were the most toxic plants in the cellular assay. These plants are toxic to animals and the cytoxicity is in line with the in vivo toxicity. The protective effects of antioxidant constituents in some extracts varied and appear to be influenced by the metabolism of the type of cell in culture. It also appears to suggest that metabolism in kidney derived cells can be influenced by species variation in the origin of cells. P. henkellii was selected for isolation of bioactive compound. Three compounds were isolated and their structure elucidated using 13C and 1H NMR and mass spectrometric data. The antibacterial, antifungal and antiviral activity of the isolated compounds 7’, 4’, 7’’, 4’’’, tetramethoxy amentoflavone (C1), isoginkgetin (C2) and Podocarpusflavone–A (C3) were determined. Compound C2 was the most active against E. coli and S. aureus (MIC = 60 ìg/mE) and a selectivity index (SI) value of 16.67. The compound was also active against A. fumigatus and C. neoformans (SI = 33.33) suggesting both antibacterial and antifungal activity with relative safety. Compound C3 had a broad spectrum of activity against E. faecalis and P. aeruginosa with SI values of 4. A less potent activity of the compounds was obtained in both the virucidal and attachment assays against test pathogens, indicating the lower activity of the compounds against tested viral pathogens. The studies further suggest structural activity relationship in the antimicrobial activity of biflavonoids. The compounds C1 and C2 had no toxic effect on the three cell types and mutagenicity studies indicated no activity of these compounds. Podocarpusflavone-A occurs in every species of Podocarpus so far investigated, except P. latifolius. These studies represent the first isolation of bioactive compounds from P. henkellii. Although a different extractant was used than that used by traditional healers, the presence of antiviral compounds in Podocarpus henkelii against two unrelated viruses may justify on a chemotaxonomic basis the traditional use of related species Podocarpus latifoliusand Podocarpus falcatus in the traditional treatment of canine distemper infection in dogs.Thesis (PhD)--University of Pretoria, 2011.Paraclinical Sciencesunrestricte

The demonstration of lumpy skin disease virus in semen of experimentally infected bulls using different diagnostic techniques

Lumpy skin disease virus (LSDV), a poxvirus that belongs to the genus Capripoxvirus is an important pathogen that can be shed in the semen of infected bulls. The screening of semen for infectious virus prior to artificial insemination requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the use of polymerase chain reaction (PCR) are sensitive diagnostic tests which can be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture is a sensitive method and detects infectious virus, its use has major limitations due to the toxic effect of semen on the cells. This study was therefore aimed at finding a method that decreases the toxic effect of semen on cell culture and enhances LSDV isolation. Secondly, the efficiency of this method in enhancing the isolation of LSDV in field samples was tested. In order to eliminate the toxic effect of semen on cell culture, a pilot study was conducted in which semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain 248/93 with a titre of 6.5 log TCID50. The semen samples were subjected to one of four different methods, viz centrifugation, serial dilution, filtration and chemical treatment with kaolin. The centrifugation, serial dilution, and filtration methods were supplemented with additional amounts of gentamycin. The toxic effects of semen on cell culture were completely eliminated when supernatants of semen samples, centrifuged at 2000 rpm for 1, 3 and 5 mins and serial diluted was used to inoculate confluent monolayers of bovine dermis cells. Semen diluted in MEM with or without additional antibiotics was the most sensitive method of demonstrating virus at higher dilutions, followed by pellets of samples centrifuged for 1 and 3 minutes. The toxicity recorded when the pellet fraction of semen samples were centrifuged for 5 mins at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. The use of filtration and kaolin treatment of semen samples could not remove the toxic effect of semen on cells. To evaluate the presence of LSDV in semen of experimentally infected bulls, six seronegative post-pubertal bulls housed in an insect proof facility were infected with LSDV via the intravenous route. The experimentally infected bulls were monitored for clinical sign of the disease. Two bulls showed severe, two a mild and two an inapparent infection. Blood samples were collected for virus isolation and semen samples for virus isolation and PCR. Vesicular fluid and preputial washes were also investigated for the presence of LSD viral nucleic acid using PCR. The infectious titre of the virus shed in semen of these bulls was also calculated. The incubation period in infected bulls varied from 7 to 14 days. The length of viraemia varied between groups and did not correlate with the severity of clinical disease. The virus was isolated from blood samples of bulls in the severely infected group on several occasions. Bulls in the mildly infected group had the lowest rate of isolated virus when compared to those with inapparent infection. The use of supernatants of centrifuged serial diluted semen samples, as shown in the pilot study, have considerably reduced the toxic effect of semen on cell culture. This method was used to test field samples for its sensitivity to isolated LSDV in semen of experimentally infected bulls with PCR as a gold standard. In all the semen samples tested using supernatants of semen samples LSDV was isolated in 53.1% of the samples on cell culture while in the serial diluted samples, only 28.1% of samples were positive with a median time of detection on cell culture of 4 and 8 days, respectively. The use of the supernatant fraction was able to detect infectious LSDV in semen samples for prolonged periods with reduced time of development of cytopathic effect, than previously reported. In order to compare the sensitivity of PCR and virus isolation, PCR positive and a few negative samples were subjected to virus isolation using the centrifugation method developed in the pilot study. The PCR was able to detect LSD viral nucleic acids in some semen samples even when virus could not be isolated on cell culture. The PCR was also able to detect viral nucleic acid in vesicular fluid and preputial washes of infected bulls. The titre of the virus shed in the semen at a certain stage of the infection was calculated to be 3 log TCID50. In conclusion, this study provides evidence of a complete reduction of the toxic effect of semen on cell culture and increase chances of LSDV isolation with reduced detection time when semen samples are processed using the centrifugation method as described in the pilot study. Furthermore, it showed PCR was more sensitive than virus isolation in the detection of LSD viral nucleic acid in semen samples and can be used for routine diagnosis. However, virus isolation must be used when the infective nature of virus shed in semen is desirable. This study provides the first evidence of the shedding of LSDV nucleic acid in vesicular fluid and preputial washes of experimentally infected bulls. It also represents the first report that a considerable amount of LSDV is shed in semen of experimentally infected bulls, which may be infective at certain stages of clinical disease. Lumpy skin disease virus (LSDV), a poxvirus that belongs to the genus Capripoxvirus is an important pathogen that can be shed in the semen of infected bulls. The screening of semen for infectious virus prior to artificial insemination requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the use of polymerase chain reaction (PCR) are sensitive diagnostic tests which can be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture is a sensitive method and detects infectious virus, its use has major limitations due to the toxic effect of semen on the cells. This study was therefore aimed at finding a method that decreases the toxic effect of semen on cell culture and enhances LSDV isolation. Secondly, the efficiency of this method in enhancing the isolation of LSDV in field samples was tested. In order to eliminate the toxic effect of semen on cell culture, a pilot study was conducted in which semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain 248/93 with a titre of 6.5 log TCID50. The semen samples were subjected to one of four different methods, viz centrifugation, serial dilution, filtration and chemical treatment with kaolin. The centrifugation, serial dilution, and filtration methods were supplemented with additional amounts of gentamycin. The toxic effects of semen on cell culture were completely eliminated when supernatants of semen samples, centrifuged at 2000 rpm for 1, 3 and 5 mins and serial diluted was used to inoculate confluent monolayers of bovine dermis cells. Semen diluted in MEM with or without additional antibiotics was the most sensitive method of demonstrating virus at higher dilutions, followed by pellets of samples centrifuged for 1 and 3 minutes. The toxicity recorded when the pellet fraction of semen samples were centrifuged for 5 mins at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. The use of filtration and kaolin treatment of semen samples could not remove the toxic effect of semen on cells. To evaluate the presence of LSDV in semen of experimentally infected bulls, six seronegative post-pubertal bulls housed in an insect proof facility were infected with LSDV via the intravenous route. The experimentally infected bulls were monitored for clinical sign of the disease. Two bulls showed severe, two a mild and two an inapparent infection. Blood samples were collected for virus isolation and semen samples for virus isolation and PCR. Vesicular fluid and preputial washes were also investigated for the presence of LSD viral nucleic acid using PCR. The infectious titre of the virus shed in semen of these bulls was also calculated. The incubation period in infected bulls varied from 7 to 14 days. The length of viraemia varied between groups and did not correlate with the severity of clinical disease. The virus was isolated from blood samples of bulls in the severely infected group on several occasions. Bulls in the mildly infected group had the lowest rate of isolated virus when compared to those with inapparent infection. The use of supernatants of centrifuged serial diluted semen samples, as shown in the pilot study, have considerably reduced the toxic effect of semen on cell culture. This method was used to test field samples for its sensitivity to isolated LSDV in semen of experimentally infected bulls with PCR as a gold standard. In all the semen samples tested using supernatants of semen samples LSDV was isolated in 53.1% of the samples on cell culture while in the serial diluted samples, only 28.1% of samples were positive with a median time of detection on cell culture of 4 and 8 days, respectively. The use of the supernatant fraction was able to detect infectious LSDV in semen samples for prolonged periods with reduced time of development of cytopathic effect, than previously reported. In order to compare the sensitivity of PCR and virus isolation, PCR positive and a few negative samples were subjected to virus isolation using the centrifugation method developed in the pilot study. The PCR was able to detect LSD viral nucleic acids in some semen samples even when virus could not be isolated on cell culture. The PCR was also able to detect viral nucleic acid in vesicular fluid and preputial washes of infected bulls. The titre of the virus shed in the semen at a certain stage of the infection was calculated to be 3 log TCID50. In conclusion, this study provides evidence of a complete reduction of the toxic effect of semen on cell culture and increase chances of LSDV isolation with reduced detection time when semen samples are processed using the centrifugation method as described in the pilot study. Furthermore, it showed PCR was more sensitive than virus isolation in the detection of LSD viral nucleic acid in semen samples and can be used for routine diagnosis. However, virus isolation must be used when the infective nature of virus shed in semen is desirable. This study provides the first evidence of the shedding of LSDV nucleic acid in vesicular fluid and preputial washes of experimentally infected bulls. It also represents the first report that a considerable amount of LSDV is shed in semen of experimentally infected bulls, which may be infective at certain stages of clinical disease.Dissertation (MSc (Veterinary Science))--University of Pretoria, 2006.Veterinary Tropical Diseasesunrestricte

Phytochemical screening, antimicrobial and cytotoxicity studies of ethanol leaf extract of Aphania senegalensis (Sapindaceae)

Background: Aphania senegalensis (Sapindaceae) is commonly used in Senegalese traditional medicine to treat pain, inflammation, asthenia, bacterial and fungal infections. The aim of this study was to determine the type of phytochemical constituents present in the ethanol leaf extract and its antimicrobial activity against selected bacterial and fungal pathogens.Materials and Methods: The ethanol leaf extract of A. senegalensis was evaluated for its cytotoxic effect in the MTT assay against Vero cells. Flavonoids and tannins were the main constituents of the ethanol leaf extract.Results: The extract inhibited the growth of the three fungal strains used in this study moderately with the lowest MIC obtained for Candida albicans (0.16 mg/mL). The extract also inhibited the growth of Aspergillus fumigatus and Cryptococcus neoformans with an MIC of 0.62 mg/mL. For bacterial pathogens, strong inhibition was obtained against Enterococcus faecalis (ATTC 29212) (MIC 0.08 mg/mL), while moderate inhibition was obtained for Escherichia coli (ATTC 25922) (MIC 0.16 mg/mL) and Staphylococcus aureus (ATTC 29213) (MIC 0.31mg/mL). The extract however did not inhibit the growth of Pseudomonas aeruginosa (ATTC 27853) at the highest concentration (2.5 mg/ml) tested. The ethanol leaf extract of A. senegalensis had a higher cytotoxicity than berberine used as the positive control (LC50 2.67±0.04 μg/mL and 9.99±0.54 μg/mL respectively). The best selectivity index values was obtained for Enterococcus faecalis (SI = 1.24), followed by Escherichia coli (SI = 0.62) for bacterial pathogens and C. albicans (SI = 0.62) for fungal pathogens.Conclusion: The findings of this study suggest that the extracts may not be safe for use in animals infected by some pathogens.Keywords: Aphania senegalensis, leaf, phytochemical, antimicrobial, cytotoxicit

Antiviral activity of six South African plants traditionally used against infections in ethnoveterinary medicine

Viral infections remain a major threat to humans and animals and there is a crucial need for new antiviral agents especially with the development of resistant viruses. The hexane, dichloromethane, acetone and methanol extracts of six plant species selected for their traditional use against infections were tested for in vitro antiviral activity against canine distemper virus (CDV), canine parainfluenza virus-2 (CPIV-2), feline herpesvirus-1 (FHV-1) and lumpy skin disease virus (LSDV). All extracts were tested for their cytotoxicity using a colorimetric tetrazolium-based (MTT) assay and were tested for antiviral efficacy at concentrations below CC50 values on the various cell types used in this study. The antiviral activity of extracts was tested using virucidal and attachment assays. In the virucidal assay, extracts were incubated with virus prior to infection. The most potent inhibition was observed with the acetone and methanol extracts of Podocarpus henkelii against CDV and LSDV, which inhibited replication of the viruses by >75% at 3 mg/ml with selectivity index (SI) values ranging between 12 and 45. Excellent activity was also found with the hexane extracts of Plumbago zeylanica and Carissa edulis against CDV, with the extracts reducing viral-induced CPE by 50% and 75% respectively. The hexane extract of C. edulis had moderate activity against FHV-1 with EC50< 70 mg/ml and SI value <2. Only the acetone extract of P. henkelii moderately inhibited replication of LSD virus in the attachment assay, with low activity in other extracts. Of the four extracts with significant antiviral activity, two were prepared from P. henkelii. Therefore, future work will focus on isolating and characterizing the substance(s) responsible for bioactivity in extracts of this speciesThe National Research Foundation and the Faculty of Veterinary Science, University of Pretoria, South Africa.http://www.elsevier.com/locate/vetmicmn201

Phytochemical screening, antimicrobial and cytotoxicity studies of ethanol leaf extract of Aphania Senegalensis (Sapindaceae)

BACKGROUND : Aphania senegalensis (Sapindaceae) is commonly used in Senegalese traditional medicine to treat pain, inflammation, asthenia, bacterial and fungal infections. The aim of this study was to determine the type of phytochemical constituents present in the ethanol leaf extract and its antimicrobial activity against selected bacterial and fungal pathogens. MATERIALS AND METHODS : The ethanol leaf extract of A. senegalensis was evaluated for its cytotoxic effect in the MTT assay against Vero cells. Flavonoids and tannins were the main constituents of the ethanol leaf extract. RESULTS : The extract inhibited the growth of the three fungal strains used in this study moderately with the lowest MIC obtained for Candida albicans (0.16 mg/mL). The extract also inhibited the growth of Aspergillus fumigatus and Cryptococcus neoformans with an MIC of 0.62 mg/mL. For bacterial pathogens, strong inhibition was obtained against Enterococcus faecalis (ATTC 29212) (MIC 0.08 mg/mL), while moderate inhibition was obtained for Escherichia coli (ATTC 25922) (MIC 0.16 mg/mL) and Staphylococcus aureus (ATTC 29213) (MIC 0.31mg/mL). The extract however did not inhibit the growth of Pseudomonas aeruginosa (ATTC 27853) at the highest concentration (2.5 mg/ml) tested. The ethanol leaf extract of A. senegalensis had a higher cytotoxicity than berberine used as the positive control (LC50 2.67±0.04 μg/mL and 9.99±0.54 μg/mL respectively). The best selectivity index values was obtained for Enterococcus faecalis (SI = 1.24), followed by Escherichia coli (SI = 0.62) for bacterial pathogens and C. albicans (SI = 0.62) for fungal pathogens. CONCLUSION : The findings of this study suggest that the extracts may not be safe for use in animals infected by some pathogens.http://journals.sfu.ca/africanem/index.php/ajtcamam2017Paraclinical Science

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

Antimicrobial activity, toxicity and selectivity index of two biflavonoids and a flavone isolated from Podocarpus henkelii (Podocarpaceae) leaves

BACKGROUND: Different parts of Podocarpus henkelii have been used in many cultures around the world to treat ailments such as cholera, stomach diseases, rheumatism, cancer, canine distemper in dogs and gall sickness in cattle. The aim of this study was to evaluate the biological activity and toxicity of isolated compounds from Podocarpus henkelii after an earlier study indicated a promising activity in crude extracts against viral pathogens of veterinary importance. METHODS: The antibacterial and antifungal activity of two biflavonoids 7, 4’, 7”, 4”’-tetramethoxy amentoflavone (TMA), isoginkgetin (IGG) and podocarpus flavone–A (PFA) isolated from the leaves of Podocarpus henkelii were determined using a serial microplate dilution method with tetrazolium violet as growth indicator. The cytotoxicity of compounds TMA and IGG were determined on different cell types using a tetrazolium-based colorimetric cellular assay (MTT). The Ames test was used to determine their mutagenic activities. RESULTS: TMA had reasonable antifungal activity against Aspergillus fumigatus (MIC = 30 μg/ml). IGG had a wide spectrum of activity against four bacterial and two fungal pathogens with much higher selectivity index values obtained for A. fumigatus and Cryptococcus neoformans (SI > 30). PFA had a broad spectrum of activity against Enterococcus faecalis and Pseudomonas aeruginosa (SI > 15) and less activity against the two fungal pathogens. In both the cytotoxicity assays and Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, TMA and IGG had no deleterious effect on the different cell types and did not induce mutations in the Ames test. CONCLUSION: Although the antimicrobial activities of the isolated compounds were not that exciting, the compounds had no cytotoxic activity at the highest concentration (1000 μg/ml) tested against all three cell lines. IGG was the most active against E. coli, S. aureus, A. fumigatus and C. neoformans, exhibiting both antibacterial and antifungal activity with good selectivity index values. PFA had a broad spectrum of activity against E. faecalis and P. aeruginosa. The two compounds isolated had low toxicity and no genotoxic activity in the Ames test.The financial support of the National Research Foundation (grant 77228) and the Faculty of Veterinary Science, University of Pretoria, South Africa is gratefully acknowledged.http://www.biomedcentral.com/bmccomplementalternmedam201

In vitro antifungal activity of the acetone extract and two isolated compounds from the weed, Pseudognaphalium luteoalbum

Invasive and weedy species such as Pseudognaphalium luteoalbum may serve as a source of biologically active extracts or compounds with application in crop and ornamental plant protection, among other uses. The acetone crude extract of P. luteoalbum leaves had strong antifungal activity when tested against a selection of plant pathogenic fungi in vitro. Fractionation of the crude extract to isolate, characterize and evaluate the antifungal constituents afforded two compounds. Structure elucidation of the isolated compounds was carried out using spectroscopic techniques: mass spectrometry and NMR (1D and 2D). The compounds were identified as: 5,4′-dihydroxy-6-methoxy-7-O-β-glucopyranosideflavone (hispidulin-7-O-glucopyranoside) (1) and stigmasterol-3-O-β-glucopyranoside (2). These compounds are reported from P. luteoalbum for the first time. The crude extract and isolated compounds were moderately to highly active against a selection of phytopathogenic fungal organisms with MIC values ranging from 0.02 to 1.25 mg/mL. No cytotoxicity of the isolated compounds against Vero kidney cells was observed at 200 μg/mL, the highest concentration tested.National Research Foundation (NRF), grant number NRF 47346, provided funding in the Southern Africa Regional Development Cooperation between Botswana and South Africa.http://www.elsevier.com/locate/sajbhb201

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

Antimicrobial activity and cytotoxicity of triterpenes isolated from leaves of Maytenus undata (Celastraceae)

BACKGROUND: Plants of the genus Maytenus belong to the family Celastraceae and are widely used in folk medicine as anti-tumour, anti-asthmatic, analgesic, anti-inflammatory, antimicrobial and anti-ulcer agents, and as a treatment for stomach problems. The aim of this study was to isolate and identify active compounds with antifungal activity from Maytenus undata after a preliminary study highlighted promising activity in crude extracts. METHODS: Sequential extracts of M. undata leaves prepared using hexane, dichloromethane (DCM), acetone and methanol (MeOH) were tested for activity against Cryptococcus neoformans, a fungal organism implicated in opportunistic infections. Bioassay-guided fractionation of the hexane extract using C. neoformans as test organism was carried out to isolate antifungal compounds. The cytotoxicity of compounds isolated in sufficient quantities was evaluated using a tetrazolium-based colorimetric cellular assay (MTT) and a haemagglutination assay (HA). RESULTS: The hexane extract was most active with an MIC of 20 μg/ml against C. neoformans. The triterpene compounds friedelin (1), epifriedelanol (2), taraxerol (3), 3-oxo-11α-methoxyolean-12-ene-30-oic acid (4), 3-oxo-11α- hydroxyolean-12-ene-30-oic acid (5) and 3,11-dihydroxyolean-12-ene-30-oic acid (6) were isolated. Compound 6 was isolated for the first time from a plant species. The antimicrobial activity of compounds 1, 3, 5 and 6 was determined against a range of bacteria and fungi implicated in opportunistic and nosocomial infections. Compounds 5 and 6 were the most active against all the tested microorganisms with MIC values ranging between 24 and 63 μg/ml, except against Staphylococcus aureus which was relatively resistant. Compounds 1 and 3 had a low toxicity with an LC50 > 200 μg/ml towards Vero cells in the MTT assay. Compounds 5 and 6 were toxic with LC50 values of 6.03±0.02 and 2.98±0.01 μg/ml, respectively. Compounds 1 and 3 similarly were not toxic to the red blood cells (RBCs) but compounds 5 and 6 were toxic, showing HA titer values of 1.33 and 0.67 respectively. CONCLUSIONS: Compounds 5 and 6 were the most active but were also relatively cytotoxic to monkey kidney cells and red blood cells, while the other isolated compounds were less active and less cytotoxic.http://www.biomedcentral.com/1472-6882/13/111am2014mn201