Cytotoxicity testing of aflatoxin (AF) on the viability of cells grown in cultures can be widely ... more Cytotoxicity testing of aflatoxin (AF) on the viability of cells grown in cultures can be widely used to predict the potential toxic effects of AF in animals. To this end, an in vitro experimental study was conducted to ascertain the toxic effects of AF extracts obtained from compound feeds in South Africa on human lymphocytes in comparison to that of an AFB 1 standard. The approach adopted was on the basis of viable cells reducing methyl tetrazolium bromide (MTT) from blue to a purple formazan dye, which was then spectrophotometrically quantified to provide the rate of cytotoxicity. Data obtained indicated no cytotoxic response in control cells, as the viability of cells without treatment with AF standard or methanolic extracts of AF extracts (negative control) using methanol as the reconstituting solvent, was 99.9% after 24 hrs of incubation. However, cell viability significantly (p<0.001) decreased upon exposure to AF extracts especially for poultry feed. This was influenced by both the dose and duration of exposure, which was much more pronounced when the cells were exposed to AFB 1 standard than for all the AF extracts tested. This implies that these feeds on exposure to AF can greatly influence animal health with respect to both the contamination dose and exposure time.
Cytotoxicity testing of aflatoxin (AF) on the viability of cells grown in cultures can be widely ... more Cytotoxicity testing of aflatoxin (AF) on the viability of cells grown in cultures can be widely used to predict the potential toxic effects of AF in animals. To this end, an in vitroexperimental study was conducted to ascertain the toxic effects of AF extracts obtained from compound feeds in South Africa on human lymphocytes in comparison to that of an AFB1 standard. The approach adopted was on the basis of viable cells reducing methyl tetrazolium bromide (MTT) from blue to a purple formazan dye, which was then spectrophotometrically quantified to provide the rate of cytotoxicity. Data obtained indicated no cytotoxic response in control cells, as the viability of cells without treatment with AF standard or methanolic extracts of AF extracts [negative control] using methanol as the reconstituting solvent, was 99.9% after 24 hrs of incubation. However, cell viability significantly (p<0.001) decreased upon exposure to AF extracts especially for poultry feed. This was influenced by both the dose and duration of exposure, which was much more pronounced when the cells were exposed to AFB1 standard than for all the AF extracts tested. This implies that these feeds on exposure to AF can greatly influence animal health with respect to both the contamination dose and exposure time.
Isolation of filamentous species of two Aspergillum genera from compound feeds produced in South ... more Isolation of filamentous species of two Aspergillum genera from compound feeds produced in South Africa, and subsequent extraction of their individual DNA in this study, presents a simple but rapid molecular procedure for high through-put analysis of the individual morphological forms. DNA was successfully isolated from the Aspergillus spp. from agar cultures by use of a commercial kit. Agarose gel electrophoresis fractionation of the fungi DNA, showed distinct bands. The DNA extracted by this procedure appears to be relatively pure with a ratio absorbance at 260 and 280 nm. However, the overall morphological and molecular data indicated that 67.5 and 51.1% of feed samples were found to be contaminated with Aspergillus flavus and Aspergillus parasiticus, respectively, with poultry feed having the highest contamination mean level of 5.7 Â 105 CFU/g when compared to cattle (mean: 4.0 Â 106 CFU/ g), pig (mean: 2.7 Â 104 CFU/g) and horse (1.0 Â 102 CFU) feed. This technique presents a readily achievable, easy to use method in the extraction of filamentous fungal DNA and it's identification. Hence serves as an important tool towards molecular study of these organisms for routine analysis check in monitoring and improving compound feed quality against fungal contamination.
A selective analytical method based on high-performance liquid chromatography, combined with atmo... more A selective analytical method based on high-performance liquid chromatography, combined with atmospheric pressure ionisation mass spectrometry, was developed for the detection of atractyloside. The analysis was performed on an Xterra Phenyl column utilising a gradient elution profile and a mobile phase consisting of 10 mM aqueous ammonium acetate buffer-methanol-acetonitrile. The calibration curve of the method (1 ng/ml-160 microg/ml) was best described by a second order polynomial function (r2 = 0.998) but displayed good linearity in the range of 100 ng/ml-1 microg/ml (r2 = 0.999). The limit of detection for the atractyloside standard was determined and found to be 100 pg/ml and the limit of quantification of atractyloside in tuber matrix was found to be 250 pg/ml. The relative standard deviation of the method was on average below 5% (n = 8). The method was successfully applied to the analysis of Callilepis laureola tubers and unknown powdered samples for the presence of atractyloside.
Oxidative C-S and C-C bond formation with aryl and alkyl thiols was catalysed under mild conditio... more Oxidative C-S and C-C bond formation with aryl and alkyl thiols was catalysed under mild conditions in a reaction vessel open to air at pH 4.5 in the presence of a commercial laccase (Novozym 51003 or Suberase) and a co-solvent (DMF) to afford 1,4-naphthoquinone sulfides. Both monothiolation and dithiolation of the 1,4-naphthohydroquinone was accomplished with the latter being favoured. In addition, unprecedented dimerisation of monothiolated intermediates occurred via C-C coupling. These commercial laccases provide a facile and a more environmentally friendly synthetic approach to both the 1,4-naphthoquinone sulfides and the 1,4-naphthoquinone sulfide dimers.
a b s t r a c t Nuclear diamination of p-hydrobenzoquinones with aromatic and aliphatic primary a... more a b s t r a c t Nuclear diamination of p-hydrobenzoquinones with aromatic and aliphatic primary amines was catalysed by an immobilised commercial laccase, Denilite Ò II Base, from Novozymes. The amine and the p-hydro-benzoquinone was reacted under mild conditions (at room temperature and at 35 °C) in a reaction vessel open to air in the presence of laccase and a co-solvent to afford, exclusively, the diaminated p-benzoqui-none. These compounds may have potential antiallergic, antibiotic, anticancer, antifungal, antiviral and/ or 5-lipoxygenase inhibiting activity.
This study investigated the anti-Candida activity of methanol extracts from freeze-dried probioti... more This study investigated the anti-Candida activity of methanol extracts from freeze-dried probiotic cells and the isolation of some constituents in the extracts. The MIC values of the probiotic methanol cell extracts against Candida albicans ranged between 1.25 and 5mg/ml after 48 h of incubation. However, Lactococcus latics subsp. lactis strain X and Lactobacillus casei strain B extracts had an MIC of 10mg/ml after 48 h of incubation. The extracts had fungistatic rather than fungicidal activity. These extracts had a much higher antifungal activity than antifungal compounds isolated from the growth medium by many other authors. This indicates that probiotics may also release antifungal compounds in their cells that could contribute to a therapeutic effect. Lactic acid (1) and 6-O-(α-D-glucopyranosyl)-1,6-di-O-pentadecanoyl-α-D-glucopyranose a novel fatty acid derivative (2) were isolated from methanol probiotic extracts and the structure of these compounds were elucidated using NMR (1 and 2D) and mass spectrometry (MS).
Plants are constantly exposed to numerous biotic or abiotic stress factors throughout their life-... more Plants are constantly exposed to numerous biotic or abiotic stress factors throughout their life-cycle. Pathogens and pathogen-derived molecules are the best studied inducers of plant defense responses, but synthetic and naturally occurring molecules have also been used to induce various types of resistance in plants. Here, an oxime molecule, 2-isonitrosoacetophenone (INAP), related to the stress metabolite citaldoxime, was used to trigger metabolic changes in the metabolome of treated Arabidopsis thaliana plants as monitored by UHPLC-MS in conjunction with principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). The chemometric methods revealed metabolites found to be significantly present in response to the treatment. These include bioconversion products (2-keto-2-phenylacetaldoxime-glycoside and l-mandelonitrile-glycoside) as well as those of which the levels are affected by the treatment (benzoic acid and derivatives, other phenylpropanoid-derived compounds and glucosinolates). Using in planta bacterial growth evaluations, INAP treatment was furthermore found to induce an anti-microbial environment in vivo.
Isonitrosoacetophenone (INAP, 2-keto-2-phenyl-acetaldoxime) is a novel inducer of plant defense. ... more Isonitrosoacetophenone (INAP, 2-keto-2-phenyl-acetaldoxime) is a novel inducer of plant defense. Oxime functional groups are rare in natural products, but can serve as substrates depending on existing secondary pathways. Changes in the metabolomes of sorghum and tobacco cells treated with INAP were investigated and chemometric tools and multivariate statistical analysis were used to investigate the changes in metabolite distribution patterns resulting from INAP elicitation. Liquid chromatography combined with mass spectrometry (UHPLC-MS) supplied unique chemical fingerprints that were generated in response to specific metabolomic events. Principal component analysis (PCA) together with hierarchical cluster analysis (HCA) and Metabolic Trees were used for data visualization. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) and shared and unique structure (SUS) plots were exploited in parallel to reveal the changes in the metabolomes. PCA indicated that the cells responded differentially to INAP through changes in the metabolite profiles. Furthermore, HCA and Metabolic Trees showed that INAP induced metabolic perturbations in both cell lines and that homeostasis was re-established over time. OPLS-DA-based shared and unique structure (SUS) plots confirmed the results and revealed differences in the metabolites distribution patterns between tobacco and sorghum cells. Chemometric analyses of metabolomic data offers insight into changes in metabolism in response to chemical elicitation. Although similar, the response in sorghum cells was found to be more consistent and well-coordinated when compared to tobacco cells, indicative of the differences in secondary metabolism between cyanogenic and non-cyanogenic plants for oxime metabolism.
Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between differen... more Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures. Using an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof. Using this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.
A selective analytical method based on high-performance liquid chromatography, combined with atmo... more A selective analytical method based on high-performance liquid chromatography, combined with atmospheric pressure ionisation mass spectrometry, was developed for the detection of atractyloside. The analysis was performed on an Xterra Phenyl column utilising a gradient elution profile and a mobile phase consisting of 10 mM aqueous ammonium acetate buffer-methanol-acetonitrile. The calibration curve of the method (1 ng/ml-160 microg/ml) was best described by a second order polynomial function (r2 = 0.998) but displayed good linearity in the range of 100 ng/ml-1 microg/ml (r2 = 0.999). The limit of detection for the atractyloside standard was determined and found to be 100 pg/ml and the limit of quantification of atractyloside in tuber matrix was found to be 250 pg/ml. The relative standard deviation of the method was on average below 5% (n = 8). The method was successfully applied to the analysis of Callilepis laureola tubers and unknown powdered samples for the presence of atractyloside.
Cytotoxicity testing of aflatoxin (AF) on the viability of cells grown in cultures can be widely ... more Cytotoxicity testing of aflatoxin (AF) on the viability of cells grown in cultures can be widely used to predict the potential toxic effects of AF in animals. To this end, an in vitro experimental study was conducted to ascertain the toxic effects of AF extracts obtained from compound feeds in South Africa on human lymphocytes in comparison to that of an AFB 1 standard. The approach adopted was on the basis of viable cells reducing methyl tetrazolium bromide (MTT) from blue to a purple formazan dye, which was then spectrophotometrically quantified to provide the rate of cytotoxicity. Data obtained indicated no cytotoxic response in control cells, as the viability of cells without treatment with AF standard or methanolic extracts of AF extracts (negative control) using methanol as the reconstituting solvent, was 99.9% after 24 hrs of incubation. However, cell viability significantly (p<0.001) decreased upon exposure to AF extracts especially for poultry feed. This was influenced by both the dose and duration of exposure, which was much more pronounced when the cells were exposed to AFB 1 standard than for all the AF extracts tested. This implies that these feeds on exposure to AF can greatly influence animal health with respect to both the contamination dose and exposure time.
Cytotoxicity testing of aflatoxin (AF) on the viability of cells grown in cultures can be widely ... more Cytotoxicity testing of aflatoxin (AF) on the viability of cells grown in cultures can be widely used to predict the potential toxic effects of AF in animals. To this end, an in vitroexperimental study was conducted to ascertain the toxic effects of AF extracts obtained from compound feeds in South Africa on human lymphocytes in comparison to that of an AFB1 standard. The approach adopted was on the basis of viable cells reducing methyl tetrazolium bromide (MTT) from blue to a purple formazan dye, which was then spectrophotometrically quantified to provide the rate of cytotoxicity. Data obtained indicated no cytotoxic response in control cells, as the viability of cells without treatment with AF standard or methanolic extracts of AF extracts [negative control] using methanol as the reconstituting solvent, was 99.9% after 24 hrs of incubation. However, cell viability significantly (p<0.001) decreased upon exposure to AF extracts especially for poultry feed. This was influenced by both the dose and duration of exposure, which was much more pronounced when the cells were exposed to AFB1 standard than for all the AF extracts tested. This implies that these feeds on exposure to AF can greatly influence animal health with respect to both the contamination dose and exposure time.
Isolation of filamentous species of two Aspergillum genera from compound feeds produced in South ... more Isolation of filamentous species of two Aspergillum genera from compound feeds produced in South Africa, and subsequent extraction of their individual DNA in this study, presents a simple but rapid molecular procedure for high through-put analysis of the individual morphological forms. DNA was successfully isolated from the Aspergillus spp. from agar cultures by use of a commercial kit. Agarose gel electrophoresis fractionation of the fungi DNA, showed distinct bands. The DNA extracted by this procedure appears to be relatively pure with a ratio absorbance at 260 and 280 nm. However, the overall morphological and molecular data indicated that 67.5 and 51.1% of feed samples were found to be contaminated with Aspergillus flavus and Aspergillus parasiticus, respectively, with poultry feed having the highest contamination mean level of 5.7 Â 105 CFU/g when compared to cattle (mean: 4.0 Â 106 CFU/ g), pig (mean: 2.7 Â 104 CFU/g) and horse (1.0 Â 102 CFU) feed. This technique presents a readily achievable, easy to use method in the extraction of filamentous fungal DNA and it's identification. Hence serves as an important tool towards molecular study of these organisms for routine analysis check in monitoring and improving compound feed quality against fungal contamination.
A selective analytical method based on high-performance liquid chromatography, combined with atmo... more A selective analytical method based on high-performance liquid chromatography, combined with atmospheric pressure ionisation mass spectrometry, was developed for the detection of atractyloside. The analysis was performed on an Xterra Phenyl column utilising a gradient elution profile and a mobile phase consisting of 10 mM aqueous ammonium acetate buffer-methanol-acetonitrile. The calibration curve of the method (1 ng/ml-160 microg/ml) was best described by a second order polynomial function (r2 = 0.998) but displayed good linearity in the range of 100 ng/ml-1 microg/ml (r2 = 0.999). The limit of detection for the atractyloside standard was determined and found to be 100 pg/ml and the limit of quantification of atractyloside in tuber matrix was found to be 250 pg/ml. The relative standard deviation of the method was on average below 5% (n = 8). The method was successfully applied to the analysis of Callilepis laureola tubers and unknown powdered samples for the presence of atractyloside.
Oxidative C-S and C-C bond formation with aryl and alkyl thiols was catalysed under mild conditio... more Oxidative C-S and C-C bond formation with aryl and alkyl thiols was catalysed under mild conditions in a reaction vessel open to air at pH 4.5 in the presence of a commercial laccase (Novozym 51003 or Suberase) and a co-solvent (DMF) to afford 1,4-naphthoquinone sulfides. Both monothiolation and dithiolation of the 1,4-naphthohydroquinone was accomplished with the latter being favoured. In addition, unprecedented dimerisation of monothiolated intermediates occurred via C-C coupling. These commercial laccases provide a facile and a more environmentally friendly synthetic approach to both the 1,4-naphthoquinone sulfides and the 1,4-naphthoquinone sulfide dimers.
a b s t r a c t Nuclear diamination of p-hydrobenzoquinones with aromatic and aliphatic primary a... more a b s t r a c t Nuclear diamination of p-hydrobenzoquinones with aromatic and aliphatic primary amines was catalysed by an immobilised commercial laccase, Denilite Ò II Base, from Novozymes. The amine and the p-hydro-benzoquinone was reacted under mild conditions (at room temperature and at 35 °C) in a reaction vessel open to air in the presence of laccase and a co-solvent to afford, exclusively, the diaminated p-benzoqui-none. These compounds may have potential antiallergic, antibiotic, anticancer, antifungal, antiviral and/ or 5-lipoxygenase inhibiting activity.
This study investigated the anti-Candida activity of methanol extracts from freeze-dried probioti... more This study investigated the anti-Candida activity of methanol extracts from freeze-dried probiotic cells and the isolation of some constituents in the extracts. The MIC values of the probiotic methanol cell extracts against Candida albicans ranged between 1.25 and 5mg/ml after 48 h of incubation. However, Lactococcus latics subsp. lactis strain X and Lactobacillus casei strain B extracts had an MIC of 10mg/ml after 48 h of incubation. The extracts had fungistatic rather than fungicidal activity. These extracts had a much higher antifungal activity than antifungal compounds isolated from the growth medium by many other authors. This indicates that probiotics may also release antifungal compounds in their cells that could contribute to a therapeutic effect. Lactic acid (1) and 6-O-(α-D-glucopyranosyl)-1,6-di-O-pentadecanoyl-α-D-glucopyranose a novel fatty acid derivative (2) were isolated from methanol probiotic extracts and the structure of these compounds were elucidated using NMR (1 and 2D) and mass spectrometry (MS).
Plants are constantly exposed to numerous biotic or abiotic stress factors throughout their life-... more Plants are constantly exposed to numerous biotic or abiotic stress factors throughout their life-cycle. Pathogens and pathogen-derived molecules are the best studied inducers of plant defense responses, but synthetic and naturally occurring molecules have also been used to induce various types of resistance in plants. Here, an oxime molecule, 2-isonitrosoacetophenone (INAP), related to the stress metabolite citaldoxime, was used to trigger metabolic changes in the metabolome of treated Arabidopsis thaliana plants as monitored by UHPLC-MS in conjunction with principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). The chemometric methods revealed metabolites found to be significantly present in response to the treatment. These include bioconversion products (2-keto-2-phenylacetaldoxime-glycoside and l-mandelonitrile-glycoside) as well as those of which the levels are affected by the treatment (benzoic acid and derivatives, other phenylpropanoid-derived compounds and glucosinolates). Using in planta bacterial growth evaluations, INAP treatment was furthermore found to induce an anti-microbial environment in vivo.
Isonitrosoacetophenone (INAP, 2-keto-2-phenyl-acetaldoxime) is a novel inducer of plant defense. ... more Isonitrosoacetophenone (INAP, 2-keto-2-phenyl-acetaldoxime) is a novel inducer of plant defense. Oxime functional groups are rare in natural products, but can serve as substrates depending on existing secondary pathways. Changes in the metabolomes of sorghum and tobacco cells treated with INAP were investigated and chemometric tools and multivariate statistical analysis were used to investigate the changes in metabolite distribution patterns resulting from INAP elicitation. Liquid chromatography combined with mass spectrometry (UHPLC-MS) supplied unique chemical fingerprints that were generated in response to specific metabolomic events. Principal component analysis (PCA) together with hierarchical cluster analysis (HCA) and Metabolic Trees were used for data visualization. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) and shared and unique structure (SUS) plots were exploited in parallel to reveal the changes in the metabolomes. PCA indicated that the cells responded differentially to INAP through changes in the metabolite profiles. Furthermore, HCA and Metabolic Trees showed that INAP induced metabolic perturbations in both cell lines and that homeostasis was re-established over time. OPLS-DA-based shared and unique structure (SUS) plots confirmed the results and revealed differences in the metabolites distribution patterns between tobacco and sorghum cells. Chemometric analyses of metabolomic data offers insight into changes in metabolism in response to chemical elicitation. Although similar, the response in sorghum cells was found to be more consistent and well-coordinated when compared to tobacco cells, indicative of the differences in secondary metabolism between cyanogenic and non-cyanogenic plants for oxime metabolism.
Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between differen... more Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures. Using an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof. Using this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.
A selective analytical method based on high-performance liquid chromatography, combined with atmo... more A selective analytical method based on high-performance liquid chromatography, combined with atmospheric pressure ionisation mass spectrometry, was developed for the detection of atractyloside. The analysis was performed on an Xterra Phenyl column utilising a gradient elution profile and a mobile phase consisting of 10 mM aqueous ammonium acetate buffer-methanol-acetonitrile. The calibration curve of the method (1 ng/ml-160 microg/ml) was best described by a second order polynomial function (r2 = 0.998) but displayed good linearity in the range of 100 ng/ml-1 microg/ml (r2 = 0.999). The limit of detection for the atractyloside standard was determined and found to be 100 pg/ml and the limit of quantification of atractyloside in tuber matrix was found to be 250 pg/ml. The relative standard deviation of the method was on average below 5% (n = 8). The method was successfully applied to the analysis of Callilepis laureola tubers and unknown powdered samples for the presence of atractyloside.
Uploads
Papers by Paul Steenkamp